Conventional Methylation-specific PCR Research Articles

A Comparison of MGMT Testing by MSP and qMSP in Paired Snap-Frozen and Formalin-Fixed Paraffin-Embedded Gliomas.

Comparative analysis of the conventional methylation-specific PCR (MSP) vs. the quantitative MSP (qMSP) assessment of the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in 34 snap-frozen (SF) glioma samples was performed. The accuracy of the semi-quantitative MSP was compared with the corresponding qMSP semi-quantitative values using two semi-quantitative cut-off values (0-unmethylated and 1-weakly methylated) to discriminate methylated from unmethylated samples. In the case of the cut-off value 0, MSP test showed 80.0% sensitivity and 78.9% specificity compared to the reference qMSP analysis. However, when using the cut-off value 1, the diagnostic accuracy of the MSP test was significantly higher (85.7% sensitivity, 85.2% specificity). Fleiss' Kappa statistical analyses indicated moderate agreement (Fleiss' Kappa Coefficient = 0.509; 70.59% agreement) between MSP and qMSP semi-quantitative measurements of MGMT promoter methylation in glioma patients, justifying the conventional MSP use in diagnostics and confirming its high reliability. Further, we aimed to compare the validity of SF and formalin-fixed paraffin-embedded (FFPE) glioma samples for MGMT testing. Statistical analyses indicated moderate overall agreement of FFPE glioma samples and SF MSP semi-quantitative measurements (Fleiss' Kappa Coefficient = 0.516/0.509; 70.0% agreement) and emphasized their low reliability in the assessment of highly methylated MGMT promoter samples.

Promoter hypermethylation of cysteine dioxygenase type 1 in patients with non-small cell lung cancer.

In the present study, promoter hypermethylation of cysteine dioxygenase type 1 (CDO1) was evaluated in non-small cell lung cancer (NSCLC) tissues to assess the value of CDO1 as a novel biomarker to improve the diagnosis of NSCLC. Tumor tissue samples and corresponding normal lung tissue samples from 42 patients with NSCLC were obtained at the Department of Thoracic Surgery, The Second Xiangya Hospital (Changsha, China). Conventional methylation-specific PCR (cMSP) and methylation-on-beads followed by quantitative methylation-specific PCR (MOB-qMSP) were used to analyze the tumor and normal lung tissue samples. Using these two methods, promoter DNA hypermethylation of the CDO1 gene was detected in 59.4 and 71.0% of tumor tissues of patients with NSCLC and in 9.4 and 0% of normal lung tissue, respectively. Compared with the rate of methylation in the well-differentiated NSCLC tissues (15.4 and 55.6%, respectively), the rate of CDO1 gene promoter methylation was higher in the poorly differentiated tissues (89.5 and 92.3%, respectively). Overall, it was demonstrated that the MOB-qMSP method had a higher positive detection rate for CDO1 hypermethylation compared with the cMSP method. In conclusion, CDO1 gene promoter hypermethylation was more frequently observed in NSCLC tissues compared with in normal lung tissues, and a high methylation frequency of the CDO1 gene in biopsy specimens of NSCLC was associated with the degree of differentiation.

Abstract A32: NISCH promoter hypermethylation, smoking and lung cancer: A case control study

Abstract Lung cancer is the most common cancer worldwide, both in terms of incidence and mortality. The high mortality of lung cancer is attributable to the presence of metastatic disease in nearly two thirds of patients at diagnosis. Detection of early stage lung cancer amenable to curative resection could potentially result in a manifold increase in survival rates. Epigenetic alterations seemingly contribute to cancer initiation and progression. Cancer specific methylation patterns of tumor suppressor genes, which precede precursor lesions, could possibly herald earlier diagnosis of lung cancer and may even have important preventive or therapeutic implications. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. NISCH codes for a nonadrenergic imidazoline-1 receptor protein nischarin that anchors to the inner layer of the plasma membrane. The orthologous mouse protein has been shown to bind alpha-5 beta-1 integrin and influence cytoskeletal organization. Forty histopathologically confirmed lung cancer cases were enrolled along with thirty smoker and thirty nonsmoker controls. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by conventional methylation specific PCR. DNA methylated using CpG methylase by the whole genome methylation protocol, was used as positive control and Nuclease free PCR grade water was used as negative control. The serum nischarin levels were estimated using commercially available ELISA kits. Statistical analysis was performed using SPSS 22.0 software. We observed that frequency of promoter hypermethylation of tumor suppressor NISCH was significantly higher in lung cancer patients and in non-cancerous smokers as compared to lifelong nonsmoker controls. This difference in methylation status of NISCH between lung cancer cases, smoker and nonsmoker controls was found to be significant (P<0.05). There was no significant association between methylation status of tumor suppressor gene NISCH and clinicopathologic variables- staging, tumor size, lymph node status, metastasis and histopathological grading. We observed a higher frequency of NISCH methylation in adenocarcinoma as compared to Squamous Cell Carcinoma. Methylation status of tumor suppressor gene NISCH was found to be significantly higher among smokers as compared to nonsmokers. But there was no significant difference in the frequency of NISCH methylation between smoker lung cancer patients and nonsmoker lung cancer patients. There was no significant difference in methylation status of NISCH with type or duration of smoking. The pack years and packs per day were significantly higher in those with methylated NISCH as compared to the unmethylated group. There was no significant association of serum nischarin levels with methylation status of NISCH, demographic variables or any clinicopathological parameters. Our findings suggest that NISCH methylation occurs in high frequencies in cfDNA from plasma of lung cancer patients. Though it was not found to correlate with stage, tumor size, lymph node status, metastases or histological grade, it was found to be significantly associated with adenocarcinomas in our study. Hence, NISCH methylation could serve as a part of blood based biomarker panel for early diagnosis of lung cancer. In addition, since NISCH is found to be highly methylated in both high risk heavy smoker controls as well as lung cancer cases irrespective of smoking status, it can be hypothesized that NISCH methylation may be the common primogenitor at which varied etiologies for lung cancer converge. Hence, in the future, it may be investigated as a universal therapeutic target for lung cancers regardless of clinicopathological heterogeneity. Citation Format: Kritika Krishnamurthy, TK Mishra, Alpana Saxena, MK Daga, Nita Khurana, Masroor Mirza. NISCH promoter hypermethylation, smoking and lung cancer: A case control study. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A32.

O10.01 * PROGNOSTIC AND PREDICTIVE BIOMARKER-BASED SUBGROUPS IN THE NOA-04 TRIAL

BACKGROUND: Molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, mutations and (consequently) loss of expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, improve prognostication and may even guide treatment decisions in patients with anaplastic gliomas. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) allow the determination of large-scale methylation profiles and genome-wide DNA copy number changes, enabling molecular subgrouping of tumors. In addition, algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation using this assay. METHODS: In the biomarker cohort of the NOA-04 trial, the diagnostic and prognostic performance of these molecular markers using single tests, HM450 data and HM450-based algorithms has been investigated to propose biological subgroups, which reflect outcomes and potentially influence treatment decisions. RESULTS: Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged progression-free survival (PFS) with chemotherapy or radiotherapy (RT), and thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, too, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. Comparisons of single assays and HM450-based algorithms revealed a high concordance for IDH and 1p/19q status. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of cases as conventional methylation-specific PCR, with 87/91 G-CIMP-positive tumors predicted as MGMT promoter-methylated. CONCLUSIONS: ATRX loss is a hallmark and favorable prognosticator of astrocytic tumors allowing a better definition of the clinically and morphologically mixed group of AOA. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wildtype, but not IDH1-mutant malignant gliomas of WHO grades III/IV. Combined IDH1/ MGMT assessment may help to individualize clinical decision making in neurooncology. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and associated with prognosis in the NOA-04 trial. HM450 arrays allowed clustering of anaplastic gliomas into relevant subgroups.

Prognostic prediction of glioblastoma by quantitative assessment of the methylation status of the entire MGMT promoter region.

BackgroundO6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is reported to be a prognostic and predictive factor of alkylating chemotherapy for glioblastoma patients. Methylation specific PCR (MSP) has been most commonly used when the methylation status of MGMT is assessed. However, technical obstacles have hampered the implementation of MSP-based diagnostic tests. We quantitatively analyzed the methylation status of the entire MGMT promoter region and applied this information for prognostic prediction using sequencing technology.MethodsBetween 1998 and 2012, the genomic DNA of 85 tumor samples from newly diagnosed glioblastoma patients was subjected to bisulfite treatment and subdivided into a training set, consisting of fifty-three samples, and a test set, consisting of thirty-two samples. The training set was analyzed by deep Sanger sequencing with a sequencing coverage of up to 96 clones per sample. This analysis quantitatively revealed the degree of methylation of each cytidine phosphate guanosine (CpG) site. Based on these data, we constructed a prognostic prediction system for glioblastoma patients using a supervised learning method. We then validated this prediction system by deep sequencing with a next-generation sequencer using a test set of 32 samples.ResultsThe methylation status of the MGMT promoter was correlated with progression-free survival (PFS) in our patient population in the training set. The degree of correlation differed among the CpG sites. Using the data from the top twenty CpG sites, we constructed a prediction system for overall survival (OS) and PFS. The system successfully classified patients into good and poor prognosis groups in both the training set (OS, p = 0.0381; PFS, p = 0.00122) and the test set (OS, p = 0.0476; PFS, p = 0.0376). Conventional MSP could not predict the prognosis in either of our sets. (training set: OS; p = 0.993 PFS; p = 0.113, test set: OS; p = 0.326 PFS; p = 0.342).ConclusionsThe prognostic ability of our prediction system using sequencing data was better than that of methylation-specific PCR (MSP). Advances in sequencing technologies will make this approach a plausible option for diagnoses based on MGMT promotor methylation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-641) contains supplementary material, which is available to authorized users.

Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial.

Molecular biomarkers including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, and O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation may improve prognostication and guide treatment decisions for patients with World Health Organization (WHO) anaplastic gliomas. At present, each marker is individually tested by distinct assays. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) enable the determination of large-scale methylation profiles and genome-wide DNA copy number changes. Algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion, and MGMT promoter methylation using a single assay. Here, we retrospectively investigated the diagnostic and prognostic performance of these algorithms in comparison to individual marker testing and patient outcome in the biomarker cohort (n = 115 patients) of the NOA-04 trial. Concordance for IDH and 1p/19q status was very high: In 92% of samples, the HM450 and reference data agreed. In discordant samples, survival analysis by Kaplan-Meier and Cox regression analyses suggested a more accurate assessment of biological phenotype by the HM450 analysis. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of samples than conventional methylation-specific PCR, with 87 of 91 G-CIMP tumors predicted as MGMT promoter-methylated. Pyrosequencing of discordant samples confirmed the HM450 assessment in 14 of 17 cases. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and are associated with prognosis in the NOA-04 trial. For MGMT, HM450 suggests promoter methylation in the vast majority of G-CIMP tumors, which is supported by pyrosequencing.

PROGNOSTIC AND PREDICTIVE BIOMARKER-BASED SUBGROUPS IN THE NOA-04 TRIAL

BACKGROUND: The WHO classification, based on morphological criteria, may be increasingly supplemented with defined molecular aberrations. These might help to resolve the discrepancy between classification and clinical outcome. Molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, mutations and (consequently) loss of expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, improve prognostication and may even guide treatment decisions in patients with anaplastic gliomas. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) allow the determination of large-scale methylation profiles and genome-wide DNA copy number changes, enabling molecular subgrouping of tumors. In addition, algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation using this assay. METHODS: In the biomarker cohort of the NOA-04 trial, the diagnostic and prognostic performance of these molecular markers using single tests, HM450 data and HM450-based algorithms has been investigated to propose biological subgroups, which reflect outcomes and potentially influence treatment decisions. RESULTS: Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged progression-free survival (PFS) with chemotherapy or radiotherapy (RT), and thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, too, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. Comparisons of single assays and HM450-based algorithms revealed a high concordance for IDH and 1p/19q status. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of cases as conventional methylation-specific PCR, with 87/91 G-CIMP-positive tumors predicted as MGMT promoter-methylated. CONCLUSIONS: ATRX loss is a hallmark and favorable prognosticator of astrocytic tumors allowing a better definition of the clinically and morphologically mixed group of AOA. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wildtype, but not IDH1-mutant malignant gliomas of WHO grades III/IV. Combined IDH1/ MGMT assessment may help to individualize clinical decision making in neurooncology. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and associated with prognosis in the NOA-04 trial. HM450 arrays allowed clustering of anaplastic gliomas into relevant subgroups. SECONDARY CATEGORY: Tumor Biology.

Abstract 43: Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing.

Abstract DNA methylation profiling of bladder cancer specific gene promoters has been shown to be a sensitive and specific diagnostic tool for early- and late-stage bladder cancer. We have recently shown that a urine-based assay that combines DNA and protein biomarkers can accurately triage patients undergoing evaluation for bladder cancer. As part of this assay, hypermethylation of Twist1 and Nid2 is performed by conventional methylation-specific PCR (cMSP). To increase throughput and lower cost, we have now developed an approach to determine the methylation at single-base resolution of CpG sites using next generation bisulfite sequencing. In addition to Twist1 and Nid2, methylation of the Vimentin promoter, which has been shown to be associated with epithelial to mesenchymal transition (EMT) in bladder cancer, was also evaluated using this technique. To determine the sensitivity of this approach, amplification was performed on control bisulfite converted genomic DNA that was either enzymatically methylated or chemically unmethylated at CpG sites using non-methylation specific PCR. Twist1, Nid2 and Vimentin PCR products were subsequently used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Methylation status was determined by aligning sequencing reads to unmethylated reference sequences. In this assay, 0.4% methylated DNA could be reliably detected in 10 ng of bisulfite converted DNA, more than doubling the sensitivity of the current assay as well as potentially generating a single-base resolution mapping of additional CpG sites not yet quantified for Twist1, Nid2 and Vimentin. The clinical performance of the Next-Gen bisulfite sequencing assays for Twist1, Nid2 and Vimentin is currently being tested using urine samples from patients with bladder cancer as well as from age matched individuals who present with hematuria but who are found to be cancer-free. The technique we have developed could be used to detect other hypermethylated genes in a variety of bodily fluids where percent methylation may be much lower than in tumor tissue samples. Citation Format: Andrew R. Dunn, Shuqiang Li, Cecilia A. Fernandez, Anthony P. Shuber. Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2013-43

Methylation of the RASSF1A promoter is predictive of poor outcome among patients with Wilms tumor

Wilms tumor (WT) has a survival rate of 90% following multimodality therapy. Nevertheless, there are some groups of patients with event-free survival rates less than 75%. In addition to clinical prognostic factors, loss of heterozygosity at 1p and/or 16q has been used to determine treatment intensity. However, the incidence of this abnormality is low, and new biomarkers are still needed. We analyzed methylation status of three tumor suppressor genes; Ras-association domain family 1 protein, isoform A (RASSF1A), DCR2, and CASP8, in 84 WTs using conventional methylation-specific PCR (cMSP), and the results were correlated with outcome. Furthermore, we analyzed the methylation status of RASSF1A by quantitative MSP (qMSP) in 171 WTs, and evaluated clinical and genetic differences between the methylated and unmethylated tumors. RASSF1A was the most frequently methylated gene identified by cMSP, and associated with a poor outcome. Patients with a RASSF1A-methylated tumor had shorter overall and event-free survival periods (P = 0.043 and 0.018, respectively), when a cut-off value of 7% by qMSP was used. The methylation was more frequent in tumors of older children than younger children (P < 0.001), and in advanced-stage tumors than early stage tumors (P = 0.001). However, multivariate analysis could not confirm the prognostic significance of RASSF1A methylation, possibly because of a small number of advanced stage tumors examined. RASSF1A methylation was correlated with LOH at 1p and/or 16q (P = 0.017), but not with WT1 abnormality, suggesting the methylation and LOH to involve the same tumorigenic pathway. The methylation status of RASSF1A might be a novel biomarker to predict outcome of WT patients.

MGMT CpG island is invariably methylated in adult astrocytic and oligodendroglial tumors with IDH1 or IDH2 mutations

We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.

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.

Abstract 4905: Quantitative high resolution detection of O6-MGMT methylation in routinely processed human glioblastoma specimens

Abstract Hypermethylation of the DNA repair gene 06-Methylguanosinemethyltransferase (06-MGMT) is an important prognostic and predictive marker for the management of glioblastoma patients. However, assessment of O6-MGMT gene methylation is not yet standardized and a comprehensive comparison of different methods described in the literature is missing. Therefore, the methylation status of the MGMT gene was analyzed in a large series of glioblastoma specimens, WHO grade IV (n = 92) using nested conventional methylation-specific PCR (MSP), real-time PCR using either SybrGreen or a sequence specific probe for detection and pyrosequencing. All specimens were routinely processed formalin-fixed paraffin-embedded tumor biopsies. For a subset of samples (n = 30) three different protocols for the treatment of genomic DNA with bisulfite were compared. As a control, 20 brain biopsies from 10 patients without glioblastoma were analyzed. MGMT mRNA level was measured using quantitative real-time RT-PCR in n = 39 samples and the mRNA expression levels were correlated to the methylation status of these samples. The three different bisulfite protocols did not show statistically significant differences concerning quality and yield of recovered DNA. A region in the MGMT promoter in which many published primers bind, displayed constitutively elevated methylation levels also in healthy brain tissue samples, thereby leading to false-positivity under many circumstances. Nested MSP and pyrosequencing correlate fairly well in scoring samples as “methylated” versus “unmethylated”, but a considerable number of samples scored “methylated” by nested MSP (n = 7) displayed only background methylation when analyzed by pyrosequencing. Only methylation levels measured by pyrosequencing correlated with mRNA expression levels (r2 = 0.32, p = 0.0002). Also, threshold definition was much more straightforward and easier to standardize using pyrosequencing. Real-time PCR based methods did not correlate very well with nested PCR or pyrosequencing. In conclusion, pyrosequencing is the most reliable methodology for methylation analysis of O6-MGMT gene methylation analysis in routinely processed glioblastoma specimens. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4905.

Immunoprecipitation of nucleosomal DNA is a novel procedure to improve the sensitivity of serum screening for the p16 hypermethylation associated with colon cancer

Background: We developed a novel method of methylation-specific PCR (MSP) using immunoprecipitation with anti-histone antibody (IP-MSP) to efficiently detect serum methylated DNA tightly bound to de-acetylated histones. Materials and methods: The detection limit of IP-MSP for p16 methylation was determined with a standard made by cell line (SKCO-1) lysate. p16 methylation of tumor and/or serum of 51 colorectal cancers and 10 adenoma patients, and 10 healthy volunteers was detected with conventional MSP or IP-MSP. Results: IP-MSP detected p16 methylation from 0.5 pg/μl of the cell lysate. The sensitivity of IP-MSP for detecting serum p16 methylation in 27 patients with tumors characterized by p16 methylation was significantly higher than that with conventional method (81% versus 59%), particularly in Stage II patients (91% versus 45%). IP-MSP detected no p16 hypermethylation in sera of adenoma patients and volunteers. Conclusions: IP-MSP is thus considered to be a promising procedure to detect serum methylated DNA in colorectal cancer patients.

Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection.

DNA methylation is an epigenetic modification that occurs at 5′ cytosine in CpG dinucleotides and is observed in high frequency in the transcription promoter regions of tumor-suppressor genes.[1,2] Such changes account for the loss of function of tumor-suppressor genes for almost every type of cancer;[3] and more genes are inactivated by promoter hyper-methylation than by genetic mutations.[4] These abnormal epigenetic changes appear to be an early event that precedes the occurrence of genetic mutations.[5-8] Hence, detecting DNA promoter methylation has significant clinical and prognostic implications for early cancer detection. Methylation-specific PCR (MSP) remains one of the most widely used methods for the sensitive detection of DNA methylation,[9] but requires gel electrophoresis and is limited to qualitative analysis. Fluorescence-based MSP variants[10,11] utilize fluorescent probes for real-time detection, thereby facilitating high-throughput and quantitative detection of DNA methylation. In recent years, quantum dots (QDs) have emerged as promising fluorophores for biological sensing due to their unique spectroscopic properties, such as broad excitation spectra, narrow size-tunable emission wavelengths, and high photostability. QDs have also been used as FRET donors to detect biomolecular targets.[12-15] The large Stokes shift and narrow emission spectra of QDs allow for FRET with a significantly reduced acceptor excitation and spectral crosstalk, and thus permits the design of QD-FRET biosensors with the minimal background necessary for detecting targets at low-concentration.[13,16] Recently, this feature of low intrinsic background of QD-FRET facilitated the development of a methylation-detection method that achieves greater sensitivity over conventional MSP methods, as exemplified by analyzing sputum samples from lung cancer patients.[16]

Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2'-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer. Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes. Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P<0.0001, Fisher's exact test). This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.

The methylation status of RASSF1A promoter predicts responsiveness to chemotherapy and eventual cure in hepatoblastoma patients

Despite the progress of therapy, outcomes of advanced hepatoblastoma patients who are refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the mortality rate, novel prognostic markers are needed for better therapy planning. We examined the methylation status of 13 candidate tumor suppressor genes in 20 hepatoblastoma tumors by conventional methylation-specific PCR (MSP) and found hypermethylation in 3 of the 13 genes. We analyzed the methylation status of these 3 genes (RASSF1A, SOCS1 and CASP8) in 97 tumors and found hypermethylation in 30.9, 33.0 and 15.5%, respectively. Univariate analysis showed that only the methylation status of RASSF1A but not the other 2 genes predicted the outcome, and multivariate analysis showed a weak contribution of RASSF1A methylation to overall survival. Using quantitative MSP, we found RASSF1A methylation in 44.3% of the 97 tumors. CTNNB1 mutation was detected in 67.0% of the 97 tumors. While univariate analysis demonstrated RASSF1A methylation, CTNNB1 mutation and other clinicopathological variables as prognostic factors, multivariate analysis identified RASSF1A methylation (p = 0.043; relative risk 9.39) and the disease stage (p = 0.002; relative risk 7.67) but not CTNNB1 mutation as independent prognostic factors. In survival analysis of 33 patients in stage 3B or 4, patients with unmethylated tumor had better overall survival than those with methylated tumor (p = 0.035). RASSF1A methylation may be a promising molecular-genetic marker to predict the treatment outcome and may be used to stratify patients when clinical trials are carried out.

Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer

DNA methylation has emerged as a promising biomarker for prostate cancer detection. In this report, we screened 36 candidate genes generated by a bioinformatic analysis of the human genome, and found that the melanoma cell adhesion molecule (MCAM) was an excellent candidate for cancer-specific methylation in prostate cancer. Direct sequencing of bisulfite-treated genomic DNA, conventional methylation-specific PCR (MSP), real-time quantitative methylation-specific PCR, immunohistochemistry, colony formation assay, and statistical analysis. We found that the melanoma cell adhesion molecule (MCAM) gene promoter was specifically methylated in prostate cancer cell lines and primary prostate cancer (PCa) but not in non-neoplastic prostate (BPH) tissues by direct sequencing of bisulfite-treated genomic DNA and conventional methylation-specific PCR (MSP). Further analysis with quantitative MSP showed greater hypermethylation of the MCAM promoter (80%, 70/88) in primary prostate cancer compared to 12.5% (3/24) in BPH. Prostatic intraepithelial neoplasias (PIN), potential precursors of prostate carcinoma, showed an intermediate methylation rate of 23% (7/30). We further observed that MCAM promoter methylation was directly correlated with tumor stage (pT3+pT4) (P = 0.001) and Gleason score (P = 0.018) in primary prostate carcinoma. Our results suggest that MCAM promoter hypermethylation deserves further attention as a potential diagnostic prostatic DNA marker in human prostate cancer.

Detection of p57KIP2 Gene Methylation Is Useful Marker for Minimal Residual Disease in Diffuse Large B Cell Lymphoma.

Background: The t(14;18)(q32;q21) translocation, which causes a BCL2/IgH gene rearrangement, is a characteristic chromosomal abnormality found mainly in B cell lymphomas, and its detection and quantification is useful for monitoring of minimal residual disease (MRD) in follicular lymphoma (FL). However, in diffuse large B cell lymphoma (DLBCL), the frequency of this rearrangement is relatively low. We revealed that the promoter region of p57KIP2 gene is frequently methylated in B cell lymphomas. Aberrant DNA methylation in the promoter regions of genes is known as the major mechanism for inactivation of tumor suppressor genes, so the detection of the methylation is expected to be used as a promising tumor specific marker. Therefore, we investigated the possibility of p57KIP2 gene promoter methylation as a biomarker for MRD detection in B cell lymphoma, especially in DLBCL.Methods: We analyzed lymphoid cell line DNA, tumor DNA from 64 patients with DLBCL, and 4 patients with FL. The bisulfite-modified DNA was used as a template for conventional methylation-specific PCR (MSP) and real-time quantitative MSP (Q-MSP).Results: In clinical samples (tumor DNA), p57KIP2 gene methylation was detected by Q-MSP in 73% (47/64) DLBCL, and 50% (2/4) FL. Using cell line DNA, which was fully methylated in promoter region of p57KIP2 gene, we determined the detection limit of Q-MSP assay. The methylated DNA could be detected in the presence of a 1000-fold excess of unmethylated DNA by Q-MSP. We could validate the level of methylated DNA in each sample by Q-MSP evaluating the p57KIP2/b-actin ratio. The sensitivity to detect MRD in bone marrow by this method was found to be equivalent to real time quantitative PCR for BCL2/IgH major breakpoint region.Conclusion: The methylation of p57KIP2 gene is detected at high frequency in DLBCL. This biomarker is thought to be conventional and widely applicable to the detection of MRD in DLBCL.

Promoter hypermethylation of GSTP1, AR, and 14‐3‐3σ in serum of prostate cancer patients and its clinical relevance

Hypermethylation of tumor suppressor genes (TSG) is thought to play an important role in tumorigenesis of prostate cancer. The main focus of research was the detection of TSG hypermethylation in cancer tissue. Our aim was to evaluate the feasibility of detection of hypermethylated genes in serum of prostate cancer patients and its correlation with clinicopathological parameters. One hundred twenty-five serum samples from 62 patients with hormone refractory prostate cancer (HRPC), 14 patients with early disease, and 49 healthy controls were examined. After DNA extraction and sodium-bisulfite treatment, conventional methylation-specific PCR (MSP) was performed for glutathione S-transferase P1 (GSTP1), androgen receptor (AR), and 14-3-3sigma. In serum of HRCP patients, frequency of GSTP1, AR, and 14-3-3sigma hypermethylation was 32.2, 40.3, and 86.6%, respectively. In serum of patients with early disease frequency of GSTP1, AR, and 14-3-3sigma, hypermethylation was 21.4, 35.7, and 85.7%. In healthy controls, frequency of GSTP1, AR, and 14-3-3sigma hypermethylation was 0, 26.5, and 55.1%, respectively. There was a significant increase of frequency of TSG hypermethylation for GSTP1 and 14-3-3sigma in HRPC patients, in comparison with healthy controls. GSTP1 hypermethylation in HRPC patients was significantly correlated with differentiation of cancer and metastatic disease. Hypermethylation of TSG can be detected in serum of prostate cancer patients. Some hypermethylated TSG can be detected in serum of healthy controls. GSTP1 was not detectable in controls and correlated significantly with Gleason score and stage of disease. Therefore, this gene may be a promising new tool in prostate cancer diagnosis.