Detection Of Aberrant DNA Methylation Research Articles

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy.

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC=0.78 in the test set and AUC=0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

Abstract 3324: Non-invasive detection of aberrant DNA methylation in colorectal cancer by multiplex methylation specific PCR

Abstract Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide, with an increasing prevalence. The 5-year relative survival rate for advanced stages is very low, with only 14% for stage IV. However, that of the early stage of CRC is about 90%. Therefore, it is important to screen CRC in the early stage. Epigenetic alterations linked to the carcinogenesis of CRC have been shown to occur earlier and more frequently than genetic alterations in CRC. Here, our study aims to develop a CRC screening methodology by applying the multiplex methylation specific PCR (MMSP) assay to detect CRC-specific methylation biomarkers, which has been pre-selected from public databases and subsequently validated in CRC samples of the Chinese population. Experimentally, cell free DNA (cfDNA) is extracted from ~4ml peripheral blood plasma. Following bisulfite conversion of the cfDNA, MMSP were applied to amplify and detect CRC-specific methylated CpG sites within the cfDNA. From the results of 28 CRCs and 52 control specimens, a sensitivity of 85.7% and the a specificity of 92.3% was achieved. Current standard and most widely used method of detecting CRC is colonoscopy screening. However, colonoscopy requires bowel preparation, and the sedation for patients is a complex and time-consuming procedure with high cost. Additionally, this invasive method also increases the possibility of infection and complications and patient compliance still largely remains a problem. Our non-invasive MMSP assay therefore provides an alternative non-invasive screening option that is cost-effective, meeting the urgent demand of early CRC detection. In addition, because colonoscopy screening currently has low adherence in Chinese population, our method holds great potential for increasing CRC screening rate in China. Citation Format: Lili Ye, Chunting Zheng, Yuan Jie, Bin Li, Yuan Li, Kunling Hu, Liuhong Zeng, Yuying Wang, Mao Mao, Peirong Ding, Taiping Shi, Mingzhi Ye. Non-invasive detection of aberrant DNA methylation in colorectal cancer by multiplex methylation specific PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3324.

Genome-wide characterization of aberrant DNA methylation patterns and the potential clinical implications in patients with endometrial cancer

Aberrant DNA methylation has been implicated in the development of the majority of human cancers. However, the association of aberrant DNA methylation with the clinical characteristics of uterine corpus endometrial carcinoma (UCEC) has not been fully explored. We performed an integrative analysis in order to examine the genome-wide DNA methylation profile and the gene expression profile of 432 UCEC samples and 46 normal tissue samples. A total of 793 differentially methylated regions (DMRs) that were associated with 472 protein-coding genes were identified, including 622 hypermethylated DMRs and 171 hypomethylated DMRs. These DMRs were capable of differentiating UCEC from normal tissues with an accuracy of 99.07% using an unsupervised hierarchical clustering method. The genomic analysis suggested that the hypermethylated DMRs were located in CpG island regions nearer to the transcription start site (TSS) compared with the hypomethylated DMRs. Functional analysis for genes associated with DMRs revealed an enrichment of methylated genes that were involved in key cancer-related biological processes and pathways, such as cell adhesion, cell differentiation and the cAMP signaling pathway. Finally, we performed a correlation analysis of the methylation levels of DMRs and patient survival time, and identified 130 DMRs. These molecular markers could classify patients into high-risk and low-risk groups with significantly different overall survival. Taken together, the present study revealed the potential applications of the detection of aberrant DNA methylation as a valuable prognostic marker for UCEC. The current findings may aid the therapeutic exploitation of UCEC treatment strategies and improve our understanding regarding the regulation of methylation in UCEC.

The role of DNA methylation in ageing and cancer.

The aim of the present review paper is to survey the literature related to DNA methylation, and its association with cancer and ageing. The review will outline the key factors, including diet, which modulate DNA methylation. Our rationale for conducting this review is that ageing and diseases, including cancer, are often accompanied by aberrant DNA methylation, a key epigenetic process, which is crucial to the regulation of gene expression. Significantly, it has been observed that with age and certain disease states, DNA methylation status can become disrupted. For instance, a broad array of cancers are associated with promoter-specific hypermethylation and concomitant gene silencing. This review highlights that hypermethylation, and gene silencing, of the EN1 gene promoter, a crucial homeobox gene, has been detected in various forms of cancer. This has led to this region being proposed as a potential biomarker for diseases such as cancer. We conclude the review by describing a recently developed novel electrochemical method that can be used to quantify the level of methylation within the EN1 promoter and emphasise the growing trend in the use of electrochemical techniques for the detection of aberrant DNA methylation.

Epigenomic biomarkers for prognostication and diagnosis of gastrointestinal cancers.

Altered epigenetic regulation is central to many human diseases, including cancer. Over the past two decade, major advances have been made in our understanding of the role of epigenetic alterations in carcinogenesis, particularly for DNA methylation, histone modifications and non-coding RNAs. Aberrant hypermethylation of DNA at CpG islands is a well-established phenomenon that mediates transcriptional silencing of tumor suppressor genes, and it is an early event integral to gastrointestinal cancer development. As such, detection of aberrant DNA methylation is being developed as biomarkers for prognostic and diagnostic purposes in gastrointestinal cancers. Diverse tissue types are suitable for the analyses of methylated DNA, such as tumor tissues, blood, plasma, and stool, and some of these markers are already utilized in the clinical setting. Recent advances in the genome-wide epigenomic approaches are enabling the comprehensive mapping of the cancer methylome, thus providing new avenues for mining novel biomarkers for disease prognosis and diagnosis. Here, we review the current knowledge on DNA methylation biomarkers for the prognostication and non-invasive diagnosis of gastrointestinal cancers and highlight their clinical application.

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

Detection of aberrant DNA methylation of tumor suppressor genes in circulating tumor cells from a pancreatic cancer patient

Detection of aberrant DNA methylation of tumor suppressor genes in circulating tumor cells from a pancreatic cancer patient

Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm

Although aberrant DNA methylation within imprinted domains has been reported in a variety of neoplastic diseases, it remains largely uncharacterized in the context of carcinogenesis. In this study, we induced T-cell lymphoma in mice by employing a breeding scheme involving mouse strains, LSL-KrasG12D and MMTV-Cre. We then systematically surveyed imprinted domains for DNA methylation changes during tumor progression using combined bisulfite restriction analysis and NGS-based bisulfite sequencing. We detected hyper- or hypo-methylation at the imprinting control regions (ICRs) of the Dlk1, Peg10, Peg3, Grb10, and Gnas domains. These DNA methylation changes at ICRs were more prevalent and consistent than those observed at the promoter regions of well-known tumor suppressors, such as Mgmt, Fhit, and Mlh1. Thus, the changes observed at these imprinted domains are the outcome of isolated incidents affecting DNA methylation settings. Within imprinted domains, DNA methylation changes tend to be restricted to ICRs as nearby somatic differentially methylated regions and promoter regions experience no change. Furthermore, detailed analyses revealed that small cis-regulatory elements within ICRs tend to be resistant to DNA methylation changes, suggesting potential protection by unknown trans-factors. Overall, this study demonstrates that DNA methylation changes at ICRs are dynamic during carcinogenesis and advocates that detection of aberrant DNA methylation at ICRs may serve as a biomarker to enhance diagnostic procedures.

Abstract 1069: DNA methylation in circulating free DNA as a new biomarker for pancreatic cancer

Abstract Despite the clinical advancement, pancreatic cancer remains one of the most deadly cancers. Pancreatic cancers are often difficult to diagnose and there is no reliable screening biomarker for early detection of this cancer. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancers. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, detection of aberrant DNA methylation from blood samples may be a promising diagnostic tool for pancreatic cancers. First, we analyzed KRAS mutation status of 100 samples obtained by Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), which is a safe and accurate technique to perform the pancreatic tissue biopsy, and found that more than 80% of the cells in the samples harbors KRAS mutation indicating high amount of cancer cells in the specimens. Next, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the deposited DNA methylation profiles of other tumor types in the public databases, such as TCGA, and identified 8 marker genes with high frequency and specificity of DNA methylation in pancreatic cancers. We validated the DNA methylation status of these genes by bisulfite pyrosequencing and quantitative methylation specific PCR. These markers were frequently methylated in EUS-FNA samples (95% of samples are methylation positive in at least one marker gene). Now we are analyzing the DNA methylation status in pancreatic cancers and corresponding blood samples in order to identify the most effective set of markers for diagnose pancreatic cancers. Our results suggest that analysis of DNA methylation in the circulating free DNA might be a safe and potent tool for evaluating and staging pancreatic cancers. Citation Format: Keiko Shinjo, Fumiharu Ohka, Keisuke Katsushima, Akira Hatanaka, Norihisa Ichimura, Zhao Juan, Kenji Yamao, Yutaka Kondo. DNA methylation in circulating free DNA as a new biomarker for pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2015-1069

Methionine increases BDNF DNA methylation and improves memory in epilepsy.

ObjectiveTemporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus.MethodsUsing the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model.ResultsWe found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade.InterpretationOur findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE.

Alteration in global DNA methylation after bisphenol a exposure in MCF-7 cells

Abstract: Bisphenol A (BPA), as synthetic monomer used especially in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Continuous release of free BPA into food, beverages, and environment has resulted in a widespread human exposure to this chemical. Role of endocrine effects of environmental chemicals such as BPA on the changes in gene expression may be associated with epigenetic mechanisms. Early detection of aberrant DNA methylation, which regulates transcription of tumor suppressor genes and activation of oncogenes in carcinogenesis process, may provide powerful mechanistic insights in the toxicity of BPA. 5-hydroxymethylcytosine (5-hmC), oxidation product of 5-methylcytosine (5-mC), is considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the global 5-mC and 5-hmC after 48 and 96 h BPA exposure in mammalian breast cancer cell line (MCF-7). 50% of inhibitory concentration (IC50) value of BPA was determined as 148 μM by MTT cytotoxicity test in MCF-7 cells. We revealed decrease on the level of global 5-mC after BPA exposure (100 nM and 1 μM) for 48 and 96 h by using 5-mC Elisa kit, whereas non-significant slightly increase were observed in the levels of 5-hmC%. We suggested that global DNA methylation may be involved in BPA toxicity in breast cancer cells. Furthermore, evaluation on the expression of enzymes involved in DNA methylation and demethylation after BPA exposure should be investigated in cell cultures. Key words: Bisphenol A, 5-methylcytosine, 5-hydroxymethylcytosine, Cytotoxicity, MCF-7 cells.

Abstract 416: New technology to detect DNA methylation in plasma sample from lung cancer patients

Abstract Epigenetic alterations have emerged as a common hallmark of many types of human cancers including lung cancers. Therefore, detection of aberrant DNA methylation from blood samples may be a promising diagnostic tool for cancers. We have been developing a highly sensitive plasma-based approach to detect aberrant DNA methylation in lung cancer patients. First, we identified seven markers from genome-wide methylation analysis, which can diagnose lung cancer in tissue samples with high accuracy (sensitivity, 91%; specificity, 100%). Next, we evaluated DNA methylation status in tissue samples and corresponding plasma samples in each patient using pyrosequencing analysis and qMSP. However, DNA methylation status between tissue and plasma samples were not concordant by both methods (pyrosequencing, 0/19 cases; qMSP, 3/9 cases). Given the concentration of DNA in plasma originated from cancerous tissue might theoretically be very low, we need to use more sensitive and accurate methods. We used digital PCR and next generation sequencer to analyze the methylation status of plasma DNA. These methods are both highly sensitive. Now we invent a new technology using specific probes, which can recognize sequence information including methylated cytosine even in one DNA molecule. This new technology might be applicable as a practical tool for lung cancer diagnosis. Citation Format: Keiko Shinjo, Eisaku Kondo, Yutaka Kondo. New technology to detect DNA methylation in plasma sample from lung cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2014-416

Exploring and Exploiting the Aberrant DNA Methylation Profile of Endocrine-Resistant Breast Cancer

we consider how exploring the DNA methylation profile of endocrine-resistant cancer could potentially provide clinically relevant biomarkers to guide new approaches to detection and therapy, as well as advance our understanding of the mechanisms underlying this disease phenotype. It is well established that profound alterations to the genome-wide DNA methylation landscape occur in early stages of cancer initiation, during cancer progression and also throughout the acquisition of drug resistance [6,7]. These alterations are characterized by global DNA hypomethylation, which promotes genome instability and oncogene activation, and local DNA hypermethylation, typically at CpG island promoters of tumor suppressor genes, which is associated with gene silencing. The detection of aberrant DNA methylation has emerged as the most promising and best developed class of epigenetic biomarkers for cancer detection [8]. Differentially methylated regions (DMRs) of DNA have been successfully characterized as biomarkers for early detection of disease, tumor classification and response to treatment in numerous cancer subtypes (as reviewed in [8,9]). The major advantage of DNA methylation biomarkers is that DNA is inherently stable and can be obtained from numerous sources including tissue, plasma, saliva and urine [9,10]. In patients with metastatic breast cancer, genomic DNA fragments, most likely derived from necrotic or apoptotic cancer cells that carry cancer-specific epigenetic alterations, can be detected and isolated from serum. The concentration of cellfree circulating DNA in plasma from healthy individuals ranges from 10–20 ng/ml, which can increase up to 1000 ng/ml in patients with metastatic disease [11]. Using cell-free circulating DNA, multiple regions of cancer-specific hypermethylated DNA can be readily amplified using The steroid hormone estrogen is critical for the development and maintenance of the female reproductive system and also has a fundamental role in breast cancer pathogenesis. In 70% of all breast cancer cases, activation of the estrogen receptor (ER) drives cancer cell proliferation and subsequent tumor development [1,2]. ER-positive breast cancer subtypes (luminal A and luminal B) are associated with better overall survival compared with ER-negative subtypes (basal, HER2 and claudin), which is in part due to the widespread use of adjuvant endocrine therapy [3]. Endocrine therapies that serve to inhibit ER signaling, such as the selective ER modulator tamoxifen, have been used effectively for almost 40 years and have been proven to reduce the risk of disease recurrence [2,4]. Nevertheless, 28% of luminal A and 43% of luminal B breast cancer patients will exhibit intrinsic or acquired drug resistance and develop distant metastases up to 15 years after initial diagnosis [4]. Metastases most commonly form in the bone, brain, lungs or liver, ruling out surgical intervention for most patients. Current second-line therapeutic strategies remain limited and responses are often short-lived. As such, the median duration of survival from time of relapse is 2.2 and 1.6 years for luminal A and luminal B breast cancer patients, respectively [5]. Endocrine-resistant disease makes up almost a quarter of all breast cancer cases and represents one of the most significant obstacles in breast cancer treatment. Therefore, there is an obvious and urgent need to improve both the way ER-positive breast cancer patients are stratified as responders to endocrine therapy and how endocrine-resistant disease is managed therapeutically. Ideally, this could be achieved with robust biomarkers predictive of treatment response that, in the case of metastatic disease, could be profiled using noninvasive assays of the blood in the absence of a tumor biopsy. Here,

Abstract 4808: Detection of aberrant DNA methylation in saliva samples as a predictor of recurrence in head and neck squamous cell carcinoma patients

Abstract Aberrant DNA methylation (hypermethylation) at CpG islands within promoter regions is an epigenetic mechanism involved in the transcriptional silencing of several tumor suppressor genes. The presence of hypermethylation is commonly reported in different human cancers and might be useful as a molecular marker for tumor cell detection and for early detection of tumor recurrence. Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent types of cancer and the lack of early detection and the high incidence of locoregional recurrence lead to high mortality rates. Therefore, the identification of new molecular markers to be used as diagnostic and prognostic indicators is of paramount importance in reducing these recurrence rates. The aim of this study was to evaluate promoter hypermethylation of tumor suppressor genes using Quantitative Methylation-Specific PCR (Q-MSP) in tumor and salivary rinse samples as a mean to detect epigenetic alterations specific to HNSCC. Tumor samples were collected from 150 patients at the time of surgery while salivary rinses were collected before and after the last curative treatment and during the follow up (every 6 months for 2 years). The hypermethylation profile of 24 genes was analyzed and allowed the selection of a panel of 5 specific and sensitive genes. At least one of these genes was found methylated in 61% of the salivary rinse samples collected before treatment, suggesting that this panel could be useful as a diagnostic tool. Patients harboring aberrant DNA methylation of these genes in the post-treatment salivary rinse showed worse 3-year overall survival (p=0.035) and 3-year free from local recurrence survival (p=0.043). Thus, our results support the idea that the evaluation of gene methylation profile in saliva, a non-invasive method of sample collection, could be a good prognostic indicator of HNSCC recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4808. doi:10.1158/1538-7445.AM2011-4808

Quantitative assessment of DNA methylation for the detection of cervical neoplasia in liquid-based cytology specimens

Previously, we have shown that methylation-specific PCR (MSP) analysis of a key panel of genes may be useful as an ancillary tool for diagnosing squamous cell carcinomas (SCC) and high-grade squamous intraepithelial lesions (HSIL) in cervical scrapings. Because quantitative MSP (QMSP) is more suitable as a screening tool than conventional MSP, we investigated the diagnostic role of QMSP for the detection of SCC and HSIL in cervical scrapings. A quantitative multiplex-MSP approach was used to examine promoter methylation of five genes (APC, HIN-1, RAR-beta, RASSF1A, and Twist) in biopsy-confirmed SCC (n = 63), HSIL (n = 45), low-grade SIL (LSIL, n = 26), and negative (n = 28) liquid-based cytology samples. For four genes (HIN-1, RAR-beta, RASSF1A, and Twist), the methylation levels among four groups were significantly different (p < 0.001 for each). Methylation levels of HIN-1, RAR-beta, RASSF1A, and Twist were increased in HSIL and SCC samples, compared with either negative or LSIL samples. However, methylation levels were not significantly different between SCC and HSIL, with the exception of RASSF1A. Receiver-operating characteristic analysis demonstrated that HIN-1, RAR-beta, RASSF1A, and Twist had the ability to distinguish HSIL/SCC from LSIL/negative samples. The two-gene combination (RASSF1A/Twist) showed the best performance in distinguishing HSIL/SCC from LSIL/negative samples. The estimated specificity of this two-gene panel for detecting HSIL/SCC was 90.7%, and its sensitivity was 74.1%. These results suggest that quantitative detection of aberrant DNA methylation in cervical scrapings may be a promising high-throughput approach for the diagnosis of HSIL/SCC.

Evaluation of promoter hypermethylation detection in serum as a diagnostic tool for breast carcinoma in Korean women

Objective We have noted that quantitative multiplex-methylation specific PCR (QM-MSP) analysis of a key panel of genes may be useful as an ancillary tool for ductal carcinoma in situ (DCIS) detection in breast tissue. In this study, we investigated aberrant promoter hypermethylation of four genes as a means to detect epigenetic alterations specific to breast carcinoma in the serum of patients with DCIS and invasive ductal carcinoma (IDC). Methods Two hundred forty-three serum samples from 89 patients with IDC, 30 patients with DCIS, and 125 age-matched healthy controls were examined. After DNA extraction and sodium bisulfite treatment, QM-MSP was performed for HIN-1, RASSF1A, RAR-β, and Twist. Results Overall significant differences in methylation levels were observed for HIN-1 ( p = 0.006), RAR-β ( p < 0.001), RASSF1A ( p = 0.004), and Twist ( p < 0.001). All four genes showed significantly higher methylation frequencies in DCIS or IDC than in control subjects ( p < 0.001 for all comparisons). However, methylation frequencies were not significantly different between DCIS and IDC. In receiver-operating characteristic analysis, the two-gene combination (RAR-β/RASSF1A) showed the best performance in distinguishing DCIS/IDC from control samples. The estimated specificity of this two-gene panel for detecting DCIS/IDC was 88.8%, and its sensitivity was 94.1%. Conclusions The quantitative detection of aberrant DNA methylation in serum samples may be a promising high throughput approach for the diagnosis of breast cancer including DCIS.

Quantitative methylation-specific PCR for the detection of aberrant DNA methylation in liquid-based Pap tests

Aberrant promoter methylation of selective tumor suppressor genes has been detected in squamous intraepithelial lesions (SIL) and invasive cervical cancer. Identification of methylation profiles of genes that can distinguish high-grade SIL (HSIL) from low-grade SIL (LSIL), and cytologically negative for intraepithelial lesion or malignancy (NILM) residual liquid-based Papanicolaou (Pap) tests may be potentially useful as an ancillary test for cervical cancer screening. Using real-time quantitative methylation-specific polymerase chain reaction (PCR) (QMSP), the authors analyzed the frequency and relative level of promoter methylation for DAPK1, IGSF4, SPARC, and TFPI2 in biopsy-confirmed HSIL and LSIL, and NILM residual liquid-based Pap tests. The percentage of methylation (%M) for each gene was calculated using the reference gene, ACTB. The cumulative methylation score for each sample, defined as the sum of %M of all 4 genes, was used to analyze the genes in combination. For each gene analyzed the frequency and relative level of methylation were increased in HSIL compared with combined NILM/LSIL samples. The cumulative methylation scores were significantly higher in HSIL samples (P < .0001). Area under the receiver operating characteristic (ROC) curve (AUC) demonstrated that methylation of each gene could distinguish HSIL from NILM/LSIL samples (AUC range, 0.6-0.67; P < or = .0028). The combination of 4 genes showed improved test performance (AUC = 0.76; P < .0001). There was no significant difference in cumulative methylation in HSIL cases with histologic outcomes of cervical intraepithelial neoplasia grade 2 (CIN2) versus CIN3. There was no association between the methylation of any gene and the presence of human papillomavirus. The methylation profile of multiple genes in combination can better distinguish HSIL from combined NILM/LSIL samples. Although aberrant DNA methylation has the potential to function as a molecular biomarker of HSIL in liquid-based Pap tests, additional genes that are selectively methylated in HSIL are needed to improve the clinical performance.

Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing

It has been suggested that detection of aberrant DNA methylation in clinical specimens such as sputum or saliva may be a valuable tumour biomarker. Any clinically applicable detection technique must combine high sensitivity with high specificity. In this study we describe methylation enrichment pyrosequencing (MEP), which benefits from the high sensitivity and specificity of methylation-specific PCR (MSP) but has a second, confirmatory, pyrosequencing step. The pyrosequencing reaction is rapid, relatively inexpensive and offers significant logistical advantages over previously described validation methods. As proof of principle, we illustrate MEP using assays of p16 and cyclin A1 promoters in a methylated DNA dilution matrix and also in a clinical setting using paired saliva and oral tumour specimens. Our results confirm that mis-priming of MSP, with subsequent false positive results, can occur frequently (perhaps 10%) in assays combining high numbers of PCR cycles and low concentrations of starting DNA. In our clinical example, MEP of saliva-derived DNA was more sensitive than standard non-methylation-specific pyrosequencing as illustrated using p16 and cyclin A1 promoter methylation assays.

Quantitative Detection of Promoter Hypermethylation in Multiple Genes in the Serum of Patients with Colorectal Cancer

While promoter hypermethylation is a common molecular alteration of human colorectal cancer that could be detected in the bloodstream, we tested the feasibility of quantitative detection of aberrant DNA methylation in multiple genes in the serum samples of colorectal cancer patients. The pre-therapeutic serum samples of 49 colorectal cancer patients and 41 age-matched controls with normal colonoscopy were examined. The presence of methylated DNA in APC (adenomatous polyposis coli), hMLH1 (human MutL homolog 1), and HLTF (helicase-like transcription factor) was detected by quantitative methylation-specific PCR (MethyLight). There was a significant difference in the concentration of methylated serum DNA between cancer patients and controls for HLTF (p= 0.015) and hMLH1 (p= 0.0001) genes, but not for APC gene (p= 0.21). In total, 28 patients with colorectal cancer and 4 controls had methylated DNA detected in at least one marker, which gave a sensitivity of 57% and specificity of 90%. All patients with methylation in two methylation markers had advanced (stage III/IV) cancer (p= 0.006) and patients with methylation in at least one marker tended to have a lower probability of survival (p= 0.08). The quantitative detection of aberrant DNA methylation in serum may be a promising high-throughput approach for the noninvasive screening and monitoring of colorectal cancer.

Detection of Aberrant DNA Methylation in Acute Lymphocytic Leukemia (ALL) Using a Real-Time Polymerase Chain Reaction (PCR) Assay.

Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res 2002; 8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood 2003; 101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.