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

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?相似文献   

乳腺癌中BRCA1、GSTP1和MGMT基因甲基化检测的临床意义

背景与目的：DNA甲基化是1种调节基因表达的重要机制,在肿瘤发生、发展中起重要作用。研究表明,在肿瘤诊断、预后及疗效判断方面,DNA甲基化是1种有潜在应用价值的肿瘤标志物。本研究通过对乳腺癌中组织标本中BRCA1,GSTP1和MGMT这3种与DNA修复功能有关基因的甲基化状态进行检测,探讨其临床应用价值。方法：在106例配对的乳腺癌及癌旁组织中,运用甲基化特异性PCR法,对BRCA1、GSTP1和MGMT基因的甲基化状况进行检测,统计分析它们与乳腺癌主要临床病理特征之间的关系。结果：在乳腺癌组织标本中,BRCA1、GSTP1和MGMT的甲基化频率分别为24.5%(26/106)、29.2%(31/106)和18.9%(20/106),明显高于对应癌旁组织7.5%(8/106)、11.3%(12/106)和4.7%(5/106)的甲基化频率,差异有统计学意义(P<0.01)。在肿瘤组织和癌旁组织中,至少有1种基因发生甲基化的分别为50.9%(54/106)和19.8%(21/106),每个标本的平均甲基化数分别为0.73和0.24,差异有统计学意义(P<0.000 1)。BRCA1基因甲基化与患者的年龄及ER(-)呈显著正相关(P=0.007和0.020);MGMT基因甲基化与乳腺癌分期、组织学分级和淋巴结转移呈显著正相关(P=0.016,0.025和0.030);GSTP1基因甲基化与淋巴结转移、肿瘤大小呈显著正相关(P=0.028和0.033);同时有超过1种基因甲基化与乳腺癌较晚的病理分期及淋巴转移正相关(P=0.028和0.007)。结论：BRCA1、GSTP1和MGMT基因甲基化状况与乳腺癌临床病理特征显著相关,同时存在多个基因甲基化预示着乳腺癌具有侵袭性表型,联合检测3者的甲基化状况可能对乳腺癌预后预测具有重要价值。     

Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue.

PURPOSE: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. EXPERIMENTAL DESIGN: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. RESULTS: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P=0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. CONCLUSIONS: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.     

Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels

To screen candidate methylation markers for early detection of breast cancer and to explore the relationship between methylation and gene expression, we performed methylated-CpG island recovery assay (MIRA) combined with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous and non-cancerous samples. Gene expression was analyzed by microarrays and validated using RT-PCR. We detected 70 significantly hypermethylated genes in breast cancer tissues, including many novel hypermethylated genes such as ITGA4, NFIX, OTX2 and FGF12. Direct bisulfite sequencing showed widespread methylation occurring in intragenic regions of the WT1, PAX6 and ITGA4 genes and in the promoter region of the OTX2 gene in breast cancer tissues. COBRA assay confirmed that the WT1, OTX2 and PAX6 genes were hypermethylated in breast cancer tissues. Clustering analysis of the gene expression of 70 significantly hypermethylated genes revealed that most hypermethylated genes in breast cancer were not expressed in breast tissues. RT-PCR assay confirmed that WT1 and PITX2 were only weakly expressed in the breast cancer tissues and were not expressed in most non-cancerous breast tissues. OTX2 and PAX6 were not expressed in either breast cancer or non-cancerous tissues. In conclusion, these results will expand our knowledge of hypermethylated genes and methylation sites for early detection of breast cancer and deepen our understanding of the relationship between methylation and gene expression. The MIRA approach can screen candidate methylated genes for further clinical validation more effectively than gene expression microarray-based strategy.     

MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms.     

Estrogen receptor gene methylation in human breast tumors

DNA methylation is known to be involved in eukaryotic gene control; it may thus exert effects during development and tumorigenesis. We have examined the methylation status of the estrogen receptor (ER) gene in different human tissues. The ER gene was found to be methylated in placental tissues, but normal breast tissues exhibited a different methylation pattern. In addition, specific sites in the hormone-binding domain of the ER gene were observed to be differently methylated in different human breast tumor specimens. We did not detect, however, any association between the ER status of a tumor and ER gene methylation at these sites. Interestingly, a difference in the methylation status between normal and adjacent breast tumor tissues was observed. Thus, DNA methylation may be considered an additional molecular measure of the genetic heterogeneity in breast cancer.     

甲状腺乳头状癌中异常甲基化基因筛查

背景与目的：DNA甲基化是一种重要表观遗传学改变,在肿瘤的发病、诊断、预后评估乃至治疗上都具有重要的临床意义。甲状腺癌是临床最常见的内分泌系统恶性肿瘤,目前国内外对甲状腺癌中DNA甲基化研究相对较少。筛查在甲状腺乳头状癌中存在异常甲基化的基因。方法：收集2014年1月—2014年6月在复旦大学附属肿瘤医院头颈外科行甲状腺癌根治手术的患者的甲状腺乳头状癌组织及癌旁的正常甲状腺组织88对。通过前期高通量甲基化DNA亲和纯化测序技术（Methylcap-Seq）初步建立甲状腺乳头状癌全基因组甲基化谱式。通过生物信息学手段及甲基化特异性PCR技术（methylation specific PCR,MSP）进行初筛和复筛。结果：通过生物信息学手段分析初步选取46个基因位点进行检测,最终发现HOXD10和FOXD2基因启动子区存在异常高甲基化改变。HOXD10基因在甲状腺癌组织中44.4%呈高甲基化,而在癌旁组织中仅20.5%（P<0.001）;FOXD2基因在甲状腺癌组织中54.5%呈高甲基化,而在癌旁组织中仅38.6%（P<0.001）。其与甲状腺癌患者临床病理因素的多因素分析表明,HOXD10高甲基化改变与甲状腺癌病灶的多灶性相关（OR=3.71）;FOXD2高甲基化改变与甲状腺癌病灶外侵相关（OR=5.91）。结论：HOXD10与FOXD2基因在甲状腺乳头状癌中存在高甲基化修饰。甲状腺乳头状癌的发生、发展可能受HOXD10与FOXD2基因的甲基化修饰调控。     

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

Background: To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors.Methods: We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines.Results: Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2''-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth.Conclusion: Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.     

乳腺癌患者外周血浆中游离的肿瘤相关DNA检测及其临床价值

目的探讨外周血浆中游离DNA变异在乳腺癌早期诊断、疗效评估和复发监控中应用的可行性。方法采用甲基化特异性PCR(MSP)方法,检测84例乳腺癌患者肿瘤组织、癌旁正常腺体组织及外周血浆中游离的肿瘤相关DNA钙黏素E(E-cadherin)和APC基因启动子甲基化畸变状况,选择10例乳腺良性疾病患者的血浆作为正常对照。结果乳腺癌肿瘤组织E-cadherin和APC基因启动子甲基化畸变频率分别为52.4%和45.2%,相应外周血浆中相同的DNA变异阳性检出率分别为33.3%和31.0%。外周血浆中DNA甲基化变异与肿瘤组织的甲基化畸变状况显著相关(E- cadherin,P<0.001;APC,P=0.002)。血浆DNA E-cadherin和APC基因甲基化畸变检测的灵敏度分别为63.6%和63.2%,特异度分别为100%和95.7%。肿瘤组织及外周血浆中游离DNA甲基化畸变与临床分期、病理类型、肿块大小及受体状况无相关性(P>0.05)。癌旁正常腺体组织及健康对照血浆中均未检测到E-cadherin和APC基因甲基化变异。结论在受检乳腺癌患者中,约1/3外周血浆中可检测到与原发肿瘤相同的E-cadherin和(或)APC基因甲基化畸变,与临床分期无相关性。在乳腺癌早期诊断、疗效评估和复发监控中,检测外周血浆中游离的肿瘤相关DNA变异有一定的可行性。     

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.     

Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women.

PURPOSE: The purpose of this research was to examine the DNA methylation profiles of primary sporadic ovarian cancers and ovarian tissues from high-risk women. EXPERIMENTAL DESIGN: We analyzed the DNA methylation status of nine cancer-related genes in 49 primary ovarian tumors, 39 nonmalignant ovarian tissues obtained from 16 women with no known risk and from 23 high-risk women with a strong family history of breast and/or ovarian cancer or BRCA1 germ-line mutations, and 11 ovarian cancer cell lines, by methylation-specific PCR. RESULTS: Our findings are as follows: (a) methylation rates of four of nine genes, RASSF1A (41%), HIC1 (35%), E-cadherin (29%), and APC (18%) were significantly higher in tumors compared with controls. At least one of the four genes was methylated in 76% of the tumors; (b) a low frequency of methylation was present in nonmalignant tissues; (c) no significant differences in methylation frequencies were seen between the nonmalignant ovarian tissues from women at high-risk and those with no known risk of developing ovarian cancer; (d) methylation of the BRCA1 gene was found in 10% of sporadic tumors but in none of the samples from women with a germ-line BRCA1 mutation; and (e) ovarian cancer cell lines showed a similar frequency of methylation to ovarian tumors except for the HIC1 gene. CONCLUSIONS: Our results suggest that aberrant methylation of specific genes, including two not described previously, may be important in ovarian cancer pathogenesis but not in ovaries at risk for cancer development.     

Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues

The methylation pattern in the promoter region of p16, DAPK, MGMT and GSTP1 genes was investigated in oral cancer tissues and tumor associated adjacent tissues, using methylation specific PCR assay. The samples constituted 60 primary oral tumors and corresponding adjacent clinically and histopathologically normal mucosa, and buccal epithelial scrapings from 20 normal healthy individuals without any tobacco habits. The incidence of hypermethylation in oral tumor and adjacent mucosa for p16 gene was 66.7 and 50%, for DAPK was 68.3 and 60%, and MGMT gene was 51.7 and 26.7%, respectively. The overall hypermethylation in the three genes in the primary tumor was 86.7%, and corresponding adjacent normal mucosa tissues 76.7%. Hypermethylation was not observed in the promoter region of GSTP1 gene in either the primary tumors or the corresponding adjacent normal mucosa. Absence of aberrant methylation in the four genes was noted in buccal scrapings from normal healthy individuals with no tobacco habits. Thus, a high frequency of promoter region hypermethylation was observed in p16, DAPK and MGMT genes in oral cancer tissues as well as in corresponding adjacent normal mucosa. Our results indicate that epigenetic alteration of these genes is a frequent event in oral cancer, and is an early event observed in normal oral mucosa of the patients, indicating the critical importance of the epigenetic alteration in chewing tobacco associated oral carcinogenesis.     

Different methylation of oestrogen receptor DNA in human breast carcinomas with and without oestrogen receptor

The methylation of the human oestrogen receptor (ER) gene was analysed by restriction enzymes in normal and neoplastic human breast tissues and cell lines. CCGG sequences in regions inside the gene, which are methylated both in normal breast and in tissues that are not the target of the oestrogen, are hypomethylated in 30% of tumours, both ER+ and ER- carcinomas. Moreover, 5' sequences of the gene, which are hypomethylated in normal breast and not in tissues not the target of oestrogen, are methylated to a lower degree in ER+ carcinomas, whereas they are methylated to a greater degree in ER- carcinomas. However, the same region is equally hypomethylated in both ER+ and ER- cancer cell lines. Our results indicate that in breast carcinomas ER DNA methylation is deranged, and in cancer cell lines is different from that observed in primary tumours. Furthermore, the abnormal methylation in the 5' end seems to be related to abnormal expression, namely diffuse hypomethylation in carcinomas with high ER content and hypermethylation in carcinomas without ER. These findings support our previous hypothesis that DNA methylation could be involved in the control of ER gene expression and demonstrate that abnormal ER gene methylation is a typical feature of breast cancers.     

DNA methylation epigenotypes in breast cancer molecular subtypes

Introduction

Identification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes.

Methods

By using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis.

Results

The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors.

Conclusions

Our results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.     

Promoter methylation of leukemia inhibitory factor receptor gene in colorectal carcinoma

Aberrant methylation of gene promoters and corresponding loss of gene expression plays a critical role in the initiation and progression of colorectal cancer. An IL-6-type cytokine receptor, leukemia inhibitory factor receptor (LIFR), is a component of cell-surface receptor complexes for multifunctional cytokines such as LIF. Herein, we report that LIFR is methylated in human colon cancer. LIFR promoter was methylated in primary tumor tissues with high frequency (65%, 52/80). Quantitative methylation-specific PCR (TaqMan-MSP) demonstrated differential promoter methylation of LIFR in primary colorectal cancer tissues as compared to normal colon tissues (5%, 4/80). LIFR methylation was not detectable in 13 normal colon mucosa samples obtained from patients without cancer. The mRNA expression of LIFR was significantly down-regulated in colon cancer tissues as compared to corresponding normal tissues. A strong expression of LIFR protein was observed in all non-malignant normal and adjacent normal colon mucosa tissues whereas down-regulated LIFR protein expression was observed in primary tumors. These results demonstrate that cancer-specific methylation and a specific decrease of LIFR expression are a common inactivation event in colon cancer development.