DNA methylation in ductal carcinoma in situ of the breast

Ductal carcinoma in situ (DCIS) is a non-obligate precursor lesion of invasive carcinoma of the breast. Current prognostic markers based on histopathological examination are unable to accurately predict which DCIS cases will progress to invasive carcinoma or recur after surgical excision. Epigenetic changes have been shown to be a significant driver of tumorigenesis, and DNA methylation of specific gene promoters provides predictive and prognostic markers in many types of cancer, including invasive breast cancer. In general, the spectrum of genes that are methylated in DCIS strongly resembles that seen in invasive ductal carcinoma. The identification of specific prognostic markers in DCIS remains elusive and awaits additional work investigating a large panel of methylatable genes by using sensitive and reproducible technologies. This review critically appraises the role of methylation in DCIS and its use as a biomarker.


Introduction
Ductal carcinoma in situ (DCIS) is a malignant epithelial proliferation confi ned by myoepithelial cells and the basement membrane of breast ducts and is a non-obligate precursor to invasive carcinoma of the breast. Th e reported incidence of DCIS, once a rare diagnosis, has increased since the introduction of mammographic screening programs and has been reported to account for approximately 25% of new diagnoses of breast cancer [1].
Th e aim of DCIS treatment is to prevent progression to invasive carcinoma and subsequent potential for metastatic disease and death. DCIS is treated primarily by surgical excision, which can be in the form of breast-conserving surgery (lumpectomy) or mastectomy. Although mastectomy is considered to be curative, the recurrence rate in patients with DCIS treated with breast-conserving surgery alone has been reported to be greater than 25% over 10 years [2]. As a result, patients treated with breastconserving surgery may also receive radiotherapy and hormonal therapy. Although several large clinical trials have reported a signifi cant reduction in recurrence rates with the addition of adjuvant treatments in patients treated with breast-conserving surgery for DCIS [2][3][4][5][6][7][8], such treatments are associated with signifi cant fi nancial cost and side eff ects [9]. Since nearly 75% of DCIS cases do not recur after surgical excision [2], there is a group of low-risk DCIS patients who would not gain additional benefi t from adjuvant treatment [10]. Accurately identifying this group of patients is desirable, not only to avoid side eff ects of treatment but also to allow better allocation of limited health resources.
Unfortunately, traditional prognostic markers are not adequate to identify low-risk DCIS patients who may be spared adjuvant hormonal treatment, and currently there is a lack of strong level I or II evidence supporting the omission of adjuvant radiotherapy in selected low-risk cases [28]. Th us, novel biomarkers are urgently required to improve individual risk-profi ling and aid treatment selection. DNA methylation of a selected panel of genes represents another potential set of markers for outcome prediction that are less dependent on scrupulous handling of the biopsy after resection [29] and remain stable even in formalin-fi xed, paraffi n-embedded material.

Assessing DNA methylation
DNA methylation is the addition of a methyl group at the carbon 5 position of cytosine by the action of DNA methyltransferase enzymes. In mammalian cells, cytosine methylation occurs predominantly at CpG dinucleotides. Regions of high CpG dinucleotide density, known as CpG islands, frequently exist in the promoter region of human genes. Aberrant hypermethylation of these promoter CpG islands can result in functional silencing of genes by the recruitment of histone deacetylases, resulting in the formation of inactive chromatin. Alterations of DNA methy lation patterns are near uni versal in cancer. In particular, inactivation of tumor suppressor genes by promoter hypermethylation can be a driver of tumorigenesis.
Commonly used methodologies for DNA methylation analysis have been comprehensively reviewed elsewhere [29][30][31][32] and are summarized in Table 1. As all methodolo gies have their advantages and limitations, interpretation of methylation results requires critical consideration of the methodology used. It should be noted that some commonly used methodologies are prone to artifacts, in particular methylation-specifi c polymerase chain reaction (MSP) approaches, which require stringent primer hybridization conditions to minimize falsepositive amplifi cation. Methods that depend on restriction enzyme digestion also may give rise to false positives if digestion is incomplete.
In general, non-quantitative methods of methylation analysis should be avoided as they detect only the presence or absence of methylation regardless of the extent of methylation and will score a sample as methylated even if only a small proportion of templates are methylated. Low-level methylation means that only a small proportion of the cells being analyzed (possibly not related to the tumor) are methylated and this may not result in detectable changes in gene transcription overall. Quantitative or semi-quantitative methodologies are required to diff erentiate low-level from high-level methylation. Th ese include MethyLight and similar quanti tative MSP technologies, DNA sequencing, and methylationsensitive high-resolution melting (MS-HRM). It should also be noted that when methy lation is heterogeneous (that is, the individual CpGs within a given region show variable methylation), even quantita tive methodologies can give variable results [30].
Th e choice of the appropriate region to be analyzed is also a source of variation and can lead to major discrepancies in results between studies. In general, the best region to use for most studies is the one where DNA methylation is most closely correlated to the transcription of the gene, although this is rarely assessed.

DNA methylation in DCIS
Over the last decade, relatively few studies have specifi cally investigated DNA methylation in DCIS. Of these, most have taken a candidate gene approach, investigating genes known to be methylated or silenced in invasive breast cancers, breast cancer cell lines, or other cancer types [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. From such studies, aberrant methylation has been reported in a large variety of genes, including every pathway involved in carcinogenesis. An additional table lists these genes and their reported methylation frequencies (Additional fi le 1). Widely divergent frequencies of methylation have been reported for some genes, refl ecting not only diff erences in patient groups but also the use of diff erent non-standardized methodologies [29].
As with many biomarker studies in DCIS, studies exami ning DNA methylation have generally included only a relatively small number of cases. For methylation, the issue is further compounded since optimally only the neoplastic element should be assessed and this requires macro-dissection or micro-dissection to isolate DCIS from the surrounding tissues so as to avoid contamination. Th e amount of DNA obtainable from DCIS lesions, which are commonly small in mass, is therefore often a limiting factor in the number of cases able to be included in studies. Furthermore, not all studies have used pure DCIS cases (cases of DCIS without associated invasive carcinoma) or have combined methylation results of DCIS occurring in the context of invasive ductal carcinoma (IDC) mixed with pure DCIS cases. Studying pure DCIS cases may be critical for several reasons. Th e in situ component of mixed DCIS-IDC has been reported to be genomically similar to the invasive component [50], whereas DCIS-IDC combined and pure DCIS have been reported to be genetically distinct [51]. In addition, a lesion that morphologically resembles DCIS may be the spread of invasive carcinoma along a duct and therefore would be expected to have the same genetic and epigenetic altera tions as invasive carcinoma.

Summary of main published studies
Th e published studies (Additional fi le 1) illustrate the complexity of assessing the overall picture of DNA methylation in DCIS. Th ese studies have investigated diff erent sets of genes, used diff erent methodologies, and examined diff erent regions of the promoter. Whereas most studies have assessed methylation as either present or absent, some have reported methylation levels as a continuous variable. Th ese then employ various cutoff s to determine the frequency and correlation of aberrant methylation with clinicopathological parameters. Earlier studies specifi cally addressing DNA methylation in DCIS, or methylation changes in the pro gression to invasive carcinoma, examined mostly a single gene [33-35, 38-42,52] or at most a handful of genes [36,37]. More recently, studies have investigated panels of selected genes [46][47][48][49]53], and a small number of studies have incorporated global approaches for methylation analysis [54,55].
Several studies have shown an increase in the number of methylated genes from normal breast tissue to benign lesions to in situ carcinoma [47,49,56]. However, for most genes, methylation has been reported to occur with similar frequency in DCIS as in IDC [47,49]. Th is suggests that, in most cases, aberrant methylation occurs before the acquisition of an invasive phenotype and may not contribute to the development of invasion. Never theless, a small number of genes such as APC, CACNA1A, CDH1, FOXC1, HOXA10, MGMT, SFPR1, TFAP2A, and TWIST1 have been reported to show diff erences in either frequency or density of methylation between DCIS and invasive carcinoma (Table 2). Th is raises the possibility of using quantitative methylation of a panel of such genes to predict disease progression.
More recently, Hoque and colleagues [46] examined the methylation status of nine genes in pure DCIS lesions and mixed DCIS-IDC lesions by using quantitative methylation-specifi c polymerase chain reaction (qMSP) and chose the cutoff for aberrant methylation on the basis of receiver operating characteristic (ROC) curves. In mixed DCIS-IDC tumors, trends to higher frequencies of APC and CDH1 methylation were found in IDC compared with DCIS. APC methylation was found in 15 (38%) of 40 DCIS samples and in 24 (53%) of 45 IDC samples, and CDH1 methylation was present in 12 (31%) of 40 of DCIS samples and 21 (47%) of 45 of invasive samples.
Muggerud and colleagues [47] examined promoter methylation in pure DCIS lesions, mixed DCIS-IDC lesions, early-stage IDCs, and normal breast tissue in order to identify potential markers of DCIS progression. Th e analysis was done quantitatively by bisulfi te pyrosequencing, and aberrant hypermethylation was defi ned as methylation levels two times above the standard deviation of the average of the normal controls. Th is study identifi ed ABCB1, FOXC1, PPP2R2B, and PTEN as recurrently methylated genes in DCIS: all had been previously reported in IDC. Methylation of FOXC1 was observed to occur with greater frequency in invasive tumors (15/28, 53.6%) compared with pure DCIS (6/27, 22.2%).
An interesting fi nding of the above study [47] was reduced FOXC1 gene expression (as detected by quantitative reverse transcription-polymerase chain reaction, or qRT-PCR) relative to normal breast tissue not only when the tumor tissue was methylated but also in those tumors that were unmethylated. Th is echoes numerous other studies in which methylation of a given gene promoter is seen in a subset of tumors that are more generally silenced for that gene. Mechanisms other than methylation, especially histone modifi cations, are known to result in gene silencing, and it has been shown that gene silencing may precede DNA methylation and thus these tumors may show varying stages along the route from histone-based silencing to histone and methylationbased silencing [57].
Park and colleagues [49] investigated cases of pure DCIS, IDC, the non-malignant epithelial lesions atypical ductal hyperplasia and fl at epithelial atypia, and normal breast tissue for methylation of 15 genes by MethyLight. Th is study reported several novel methylated genes in DCIS (DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2). Although methylated genes accumulating at each step of abnormality were identifi ed, no diff erences in methylation frequencies between DCIS and IDC were found for most genes, with the exception of HOXA10, which was more frequently methylated in IDC (17/50, 34%) compared with DCIS (3/35, 9%) (P = 0.007).
Moelans and colleagues [48] investigated promoter methylation of 25 genes in mixed DCIS-IDC cases with methylation-specifi c multiplex-dependent probe amplifica tion (MS-MLPA). No diff erences in the number of methylated genes between the DCIS and invasive components were observed. Verschuur-Maes and colleagues [53] also used MS-MLPA to analyze promoter methylation of a panel of 50 genes in 15 columnar cell lesions (a benign epithelial lesion) and co-existent DCIS (n = 12) and IDC (n = 14). Whereas the number of methylated genes diff ered between normal breast tissue and lesional tissue, no statistical diff erence in the number of methylated genes was found between columnar cell lesions, DCIS, and IDC. However, MGMT and CACNA1A individually were observed to be more frequently methylated in invasive cancer compared with DCIS (MGMT methylation: 8/14 invasive, 2/12 DCIS, P = 0.022; CACNA1A methylation: 6/14 invasive, 1/12 DCIS, P = 0.048). Controversially, both studies [48,53] using MS-MLPA reported methylation of BRCA2 in a sizeable number of tumors, and this casts doubt on the specifi city of this methodology.
In some studies, methylation levels of genes have been assessed by using quantitative real-time polymerase chain reaction (qPCR). Th ese show that methylation levels are higher in IDC than DCIS. How much this refl ects tumor heterogeneity and how much this represents methodological issues in assessing increased densities of methylation remain uncertain. Signifi cantly higher SFRP1 methylation levels have been observed in IDC compared with DCIS [49]. Similarly, Muggerud and colleagues [47] found signifi cantly higher FOXC1 methyla tion levels in IDC (P = 0.007) and mixed tumors (P = 0.001) compared with pure DCIS. Higher methylation levels of MGMT (P = 0.019) [53] and CDH1 (P <0.04) [46] in IDC compared with DCIS have also been reported.
Whereas the above studies examined gene-specifi c DNA methylation at stages of breast cancer progression, Lee and colleagues [44] examined diff erences in DNA methylation in DCIS between American and Korean women. Quanti tative multiplex methylation-specifi c poly merase chain reaction (QM-MSP) was used to assess methylation of a panel of 10 genes in DCIS lesions from 52 American and 48 Korean women and normal breast tissue. Although Korean women have a markedly lower incidence of DCIS, the patterns of methylation were similar in the two groups, indicating that similar mechanisms of pathogenesis underlie DCIS in the two populations.

Global methylation studies of DCIS
Recently, studies have taken a global methylation approach to investigating DNA methylation in DCIS. Tommasi and colleagues [55] identifi ed 108 aberrantly methylated CpG islands by methylated CpG island recovery assay-assisted microarray analysis (MIRA) in early-stage breast cancer and six cases of undissected DCIS. Candidate genes were identifi ed on the basis of these methylated CpG islands, and six novel aberrantly methylated genes in DCIS (TLX1, HOXB13, HNF1B, GFI1, NR2E1, and HLXB9) were verifi ed by combined bisulfi te restriction analysis (COBRA). However, though identifi ed as novel, these were not validated in an independent cohort of DCIS to assess their signifi cance or used to examine the issue of recurrence or progression.
Another recent study used a global methylation approach to identify methylated genes on a panel of lowgrade invasive breast cancer and in situ cancer and then profi led selected novel genes against a small number of additional in situ and invasive breast cancers [54]. Th e hypermethylated regions, identifi ed by methyl-CpG immuno precipitation and human CpG island arrays, were used to select candidate genes on the basis of the extent and frequency of methylation changes and the proximity of these changes to the gene promoters. Methy lation of these selected genes was then analyzed by

DNA methylation as a predictive and prognostic marker in DCIS
In invasive breast carcinoma, the methylation status of certain genes has been reported to be associated with survival [58][59][60][61][62], risk of metastatic disease [63][64][65][66], risk of disease recurrence [62,64], and response to adjuvant treatment [60,67,68]. However, in DCIS, no direct link between aberrant methylation and risk of recurrence, risk of progression to invasive disease, or likelihood of response to adjuvant therapy has been reported. However, previous reports indicate associations between certain methylated genes and known predictive factors such as hormone receptor status and HER2 amplifi cation and adverse prognostic markers such as high nuclear grade, high proliferation index, TP53 mutations, and HER2 amplifi cation [36,44,[47][48][49] (Table 3). Although associations between methylated genes and currently known prognostic and predictive factors suggest that DNA methylation may have a role as a biomarker in DCIS, it has to be noted that these associations are the results of single studies that had relatively small numbers and that used diff erent methy lation analysis methodologies. Importantly, these studies were not designed to investigate the relationship of methy la tion with clinical outcome. Further well-powered studies on larger gene sets with detailed clinical data are required to establish the role of DNA methylation as a prognostic and predictive marker in DCIS.

Future outlook
Th ere are inherent diffi culties in conducting methylation studies in DCIS and this is due in large part to the nature of the disease itself. Pure DCIS cases are relatively rare compared with DCIS occurring in the context of invasive carcinoma, fresh tissue is almost never available, and usually only small amounts of formalin-fi xed, paraffi nembedded tissue are available as a source of DNA. In addition, the use of robust methodologies for DNA methy lation analysis is essential for the appropriate interpretation of methylation status.
Current knowledge of DNA methylation in DCIS is based largely on studies employing a candidate gene approach to methylation analysis. Global approaches, involving either high-throughput microarray-based assays such as the Infi nium platform or one of a variety of approaches using the power of massive parallel sequencing (MPS), are now required.
Th e application of MPS platforms in genome-wide methylation analysis and their relative advantages and disadvantages have been comprehensively reviewed by several authors [69][70][71][72][73]. MPS has already been used for genome-wide methylation analysis of non-small cell lung cancer tissue [74], colon cancer tissue [75], prostate cancer cell lines and tissue [76], and breast cancer cell lines [77][78][79].
In addition to the identifi cation of aberrant DNA methylation in the DCIS genome, the eff ect of methylation on gene expression and, importantly, clinical outcomes needs to be addressed. As DCIS is a heterogeneous disease with relatively few disease events occurring over decades, studies involving large numbers of pure DCIS cases with detailed clinical annotation and long-term follow-up are required to establish the validity of aberrant methylation as a predictive and prognostic biomarker in DCIS.

Conclusions
Identifying patients in whom DCIS will recur or progress to invasive carcinoma after surgical excision would allow appropriate allocation of limited health resources and avoid over-treatment of patients at low risk of further disease. DNA methylation has been found to be a predictive and prognostic marker in many forms of cancer. Although studies have shown that DNA methylation exists and may play a role in determining outcome in DCIS, we currently have an incomplete understanding of the role of DNA methylation in this disease. Studies specifi cally designed to investigate the relationship between DNA methylation and clinical outcome in DCIS are required to establish the validity of aberrant DNA methylation as a predictive and prognostic biomarker in DCIS.

Competing interests
The authors declare that they have no competing interests.