Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Montebello, Oslo, N-0310, Norway.
Breast Cancer Res. 2010;12(1):R3. doi: 10.1186/bcr2466. Epub 2010 Jan 7.
Ductal 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.
Quantitative 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.
Aberrant 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.
Quantitative 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.
导管原位癌（DCIS）是一种非浸润性乳腺病变，常通过乳房 X 线照相术检测到，随后通过手术切除。然而，据估计，大约一半被检测到的病变永远不会发展成浸润性癌。鉴定 DCIS 和浸润性癌的特异性表观遗传病变，并了解这些表观遗传变化如何参与触发肿瘤进展，对于更好地理解哪些病变有进展为浸润性的风险非常重要。
通过焦磷酸测序对 27 例纯 DCIS、28 例小浸润性导管癌（IDC）、34 例伴有 DCIS 成分的 IDC 和 5 例正常乳腺组织样本进行 ABCB1、CDKN2A/p16INK4a、ESR1、FOXC1、GSTP1、IGF2、MGMT、MLH1、PPP2R2B、PTEN 和 RASSF1A 的定量 DNA 甲基化分析。FOXC1、ABCB1、PPP2R2B 和 PTEN 在另外 23 例正常乳腺组织样本中进行了分析。进行 FOXC1 的实时 PCR 表达分析。
在所有三个诊断组中，以下基因均存在异常 DNA 甲基化：ABCB1、FOXC1、GSTP1、MGMT、MLH1、PPP2R2B、PTEN 和 RASSF1A。对于这些基因中的大多数，在 DCIS 水平上已经存在甲基化，与 IDC 中的甲基化频率相同。对于 FOXC1，在正常组织和浸润性肿瘤之间观察到甲基化水平存在显著差异（P < 0.001）。与纯 DCIS 相比，纯 IDC 和伴有 DCIS 的 IDC 的平均 DNA 甲基化水平明显更高（P = 0.007 和 P = 0.001）。对 25 例 DCIS、23 例 IDC 和 28 例正常组织样本的 FOXC1 表达的实时 PCR 分析显示，与正常组织相比，甲基化和非甲基化肿瘤中的 FOXC1 基因表达水平均较低（P < 0.001）。FOXC1、GSTP1、ABCB1 和 RASSF1A 的 DNA 甲基化水平在雌激素受体（ER）阳性肿瘤中高于 ER 阴性肿瘤；而在具有 TP53 突变的肿瘤中，FOXC1、ABCB1、PPP2R2B 和 PTEN 的甲基化水平较低。
定量甲基化分析鉴定了 ABCB1、FOXC1、PPP2R2B 和 PTEN 为 DCIS 中发生甲基化的新基因。特别是，FOXC1 在浸润性肿瘤中的甲基化频率显著增加。甲基化和非甲基化的 DCIS 和 IDC 中 FOXC1 基因表达降低表明，其表达缺失是乳腺癌进展过程中的早期事件。
