Quantitative Methylation-specific Polymerase Chain Research Articles

OS05.2.A MGMT promoter status in IDH1/2 mutant anaplastic astrocytoma patients assessed by DNA methylation profiling and qMS-PCR: a report from the EORTC Brain Tumor Group

Abstract BACKGROUND Temozolomide (TMZ) efficacy in high-grade glioma is related to O 6-methylguanine DNA methyltransferase promoter (MGMTp) methylation. We compared the prognostic and predictive effect of MGMTp between DNA methylation profiling (the MGMT-STP27 model) and quantitative methylation specific polymerase chain reaction (qMS-PCR) in isocitrate dehydrogenase 1 and 2 (IDH1/2) mutant (mt) anaplastic astrocytoma patients. MATERIAL AND METHODS The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy (RT), RT with concurrent TMZ, RT with 12 cycles of adjuvant TMZ, or RT with concurrent and adjuvant TMZ. MGMTp methylation status was assessed with the MGMT-STP27 model using 850k EPIC data, and qMS-PCR. IDH1/2 mutation status was determined with a next-generation sequencing panel. Overall survival (OS) was measured from date of randomization. RESULTS We identified 444 IDH1/2mt anaplastic astrocytoma patients of which MGMT-STP27 data was available for 440 patients (99.1%), qMS-PCR data for 361 patients (81.3%), and both for 357 patients (80.4%). MGMTp was methylated in 365 patients (83.0%) for the MGMT-STP27 model, and 168 patients (46.5%) for qMS-PCR. The agreement between the MGMT-STP27 model and qMS-PCR is 59.9% with a Cohen’s Kappa score of 0.229. At database lock, 289 patients with MGMT-STP27 data were still alive and 236 patients with qMS-PCR data. The median OS of MGMTp methylated glioma patients was 9.1 yrs [95 % confidence interval (CI) 7.5-not reached] for the MGMT-STP27 model, and not reached [95 % CI 9.1-not reached] for the qMS-PCR data. For MGMTp unmethylated glioma patients, the median OS was 6.9 yrs [95% CI 6.2-not reached] for the MGMT-STP27 model, and 6.8 yrs [95% CI 6.2–9.7] for the qMS-PCR data. The hazard ratio (HR) for OS based on MGMTp methylation was 0.88 [95% CI 0.58–1.31] for the MGMT-STP27 data, and 0.72 [95% CI 0.50–1.03]) for the qMS-PCR data. The HR for OS after RT with any TMZ vs RT alone for the MGMT-STP27 model was 0.53 [95% CI 0.37–0.78] for MGMTp methylated, and 0.54 [95% CI 0.25–1.18] for MGMTp unmethylated glioma patients; and for the MS-PCR data was 0.34 [95% CI 0.19–0.61] for MGMTp methylated, and 0.53 [95% CI 0.33–0.85] for MGMTp unmethylated glioma patients. CONCLUSION MGMTp methylation, regardless of assay, was neither prognostic nor predictive for outcome to temozolomide in IDH1/2mt anaplastic astrocytoma patients.

Real time quantitative methylation detection of PAX1 gene in cervical cancer screening

IntroductionDNA methylation is currently found to be associated with the progression of cervical intraepithelial neoplasia and the development of cervical cancer. The aim of this study was to analyze the...

Association of multiple candidate genes with mild cognitive impairment in an elderly Chinese Uygur population in Xinjiang.

BackgroundMild cognitive impairment (MCI) is a high‐risk factor for Alzheimer's disease (AD). In the present study, we investigated the association of genetic polymorphisms of five genes (8‐oxoguanine DNA glycosylase 1 (OGG1), bridging integrator 1 (BIN1), sortilin‐related receptor 1 (SORL1), presenilin 2 (PSEN2) and nerve growth factor (NGF)) with MCI risk in a Xinjiang Uygur population. We also tested the relationship between the promoter methylation of genes OGG1 and dihydrolipoamide S‐succinyltransferase (DLST) with MCI.MethodsThis study involved 43 MCI patients and 125 controls. Genotyping was done by Sanger sequencing. DNA methylation assays used quantitative methylation‐specific polymerase chain reaction.ResultsWe found that polymorphisms of five genes and the methylation of DLST and OGG1 genes were not associated with MCI (P > 0.05). Further subgroup analysis found that DLST hypomethylation was significantly associated with MCI in the carriers of apolipoprotein E (APOE) ε4 (P = 0.042). In the carriers of non‐APOE ε4, DLST methylation levels were significantly lower in the male control group than in the female control group (p = 0.04). Meanwhile, among the non‐APOE ε4 carriers younger than 75, OGG1 hypermethylation levels were significantly associated with MCI (P = 0.049). DLST methylation in female controls was significantly lower than that in male controls (P = 0.003). According to gender stratification, there was a significant positive correlation of fasting plasma glucose (FBG) and high‐density lipoprotein (HDL) with OGG1 methylation in the female controls (FBG: P = 0.024; HDL: P = 0.033). There was a significant inverse correlation between low‐density lipoprotein and DLST methylation in male MCI (P = 0.033). There was a significant positive correlation between HDL and DLST methylation levels in the female controls (P = 0.000).ConclusionsThis study was the first to discover that DLST promoter methylation interacted with APOE ε4 and thus affected the pathogenesis of MCI. In addition, OGG1 promoter methylation interacted with several other factors to increase the risk of MCI.

Prediction of onset of remnant gastric cancer by promoter DNA methylation of CDO1/HOPX/Reprimo/E-cadherin.

BackgroundEarly detection of remnant gastric cancer (RGC) is required to reduce the risk of death, but long-term endoscopic surveillance is difficult after gastrectomy. In this study, data for the methylation status of 4 methylation genes (CDO1, HOPX, Reprimo, and E-cadherin) to predict the onset of RGC are presented.ResultsThe 4 genes showed hypermethylation in RGC tumors in contrast to the corresponding non-cancerous mucosa tissues. The methylation level in the non-cancerous mucosa tissues of the initial surgery was obviously high in initial malignant disease for CDO1 (P = 0.0001), while in initial benign one for E-cadherin (P = 0.003). Promoter DNA methylation status in the remnant non-cancerous mucosa tissues together with the basic clinical data in turn predicted either initial malignant disease or initial benign disease with a high AUC score of 0.94, suggesting that methylation events are differentially recognized between the initial malignant and benign disease. We then finally confirmed that 4 genes hypermethylation of the non-cancerous tissues by biopsy prior to onset of RGC could predict terms until RGC occurred (P < 0.0001).MethodsA total of 58 RGC patients were used to establish the model. The 4 genes promoter methylation were analyzed for DNA obtained from the patient’s specimens using quantitative methylation specific polymerase chain reaction.ConclusionsThis risk model would help provide guidance for endoscopic surveillance plan of RGC after gastrectomy.

Association of OGG1 and DLST promoter methylation with Alzheimer's disease in Xinjiang population.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to progressive memory and cognitive impairment. Previous studies have identified multiple genes associated with AD. The aim of the present study was to validate the association of the five AD-associated variants, 8-oxoguanine DNA glycosylase 1 (OGG1) rs1052133, bridging integrator 1 rs744373, sortilin-related receptor 1, rs1133174, presenilin 2 rs8383, and nerve growth factor rs6330, in the Xinjiang Chinese population. In addition, the present study evaluated the contribution of the promoter methylation of two genes, OGG1 and dihydrolipoamide succinyltransferase (DLST) to the risk of AD. A total of 17 AD cases and 34 controls were recruited from Xinjiang province in China. Genotyping was done by Sanger sequencing. DNA methylation assay was performed using quantitative methylation specific polymerase chain reaction. The study was unable to repeat the previous association of the five genetic polymorphisms with AD. However, DLST methylation levels were demonstrated to be significantly decreased in AD patients (P=0.027), particularly in female AD patients (P=0.025). Subgroup analysis by apolipoprotein E (APOE ε4) genotype demonstrated that OGG1 methylation levels were significantly increased in APOE non-ε4 carriers compared with APOE ε4 carriers (P=0.027). In summary, the present study reported that DLST hypomethylation was significantly associated with AD in females, and that OGG1 promoter methylation may interact with APOE ε4 genotype.

Quantitative analysis of protocadherin 10 promoter methylation in peripheral blood of patients with gastric cancer by real-time and methylation specific polymerase techniques and its significance

Objective To investigate protocadherin 10 (PCDH10) methylation at promoter 5’-cytosine-phosphoric acid-guanine motif (CpG) island as a tool for non-invasive diagnosis and prognosis evaluation of primary gastric cancer. Methods Real-time fluorescent quantitative methylation specific polymerase chain reaction (Real-time MSP) technique was used for detection of PCDH10 promoter methylation in both tissue and serum specimens from 202 gastric cancer (GC), 52 gastric epithelial dysplasia, and 68 chronic gastritis, as well as 120 healthy human serum specimens. The relationship between PCDH10 methylation with clinicopathological parameters and prognosis was analyzed. Results The incidence of PCDH10 methylation in gastric cancer tissues (80.2%, 162/202) was significantly higher than that in paired adjacent tissues (26.7%, 54/202), gastric epithelial dysplasia (23.1%, 12/52) and chronic gastritis specimens (14.7%, 10/68) (all P=0.000). Detectable rate of PCDH10 methylation in serum specimens was in good agreement with that in the paired tumor tissues, with the Aζ value being 0.862 (P=0.000). The level of PCDH10 methylation in serum was closely correlated to age at diagnosis (P=0.018), H. pylori (Hp) infection (P=0.000), tumor size (P=0.000), tumor differentiation (P=0.002), lymphatic invasion (P=0.000), venous invasion (P=0.007), invasion depth (P=0.000), lymphatic metastasis (P=0.000), distant metastasis (P=0.015), clinical stages (P=0.000) and poor prognosis (P=0.000), but not with gender (P=0.435), tumor site (P=0.458) and growth pattern (P=0.435). Conclusion PCDH10 methylation plays an important role in the tumorigenesis and development of gastric cancer. Key words: Gastric cancer; Protocadherin 10; DNA methylation; Early diagnosis; Prognosis

DNA methylation is a common molecular alteration in colorectal cancer cells and culture method has no influence on DNA methylation.

The present study aimed to explore whether culture method had an influence on DNA methylation in colorectal cancer (CRC). In the present study, CRC cells were cultured in two-dimensional (2D), three-dimensional (3D) and mouse orthotopic transplantation (Tis) cultures. Principal component analysis (PCA) was used for global visualization of the three samples. A Venn diagram was applied for intersection and union analysis for different comparisons. The methylation condition of 5′-C-phosphate-G-3′ (CpG) location was determined using unsupervised clustering analysis. Scatter plots and histograms of the mean β values between 3D vs. 2D, 3D vs. Tis and Tis vs. 2D were constructed. In order to explore the biological function of the genes, gene ontology and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analyses were utilized. To explore the influence of culture condition on genes, quantitative methylation specific polymerase chain reaction (QMSP) was performed. The three samples connected with each other closely, as demonstrated by PCA. Venn diagram analysis indicated that some differential methylation positions were commonly shared in the three groups of samples and 16 CpG positions appeared hypermethylated in the three samples. The methylation patterns between the 3D and 2D cultures were more similar than those of 3D and Tis, and Tis and 2D. Results of gene ontology demonstrated that differentially expressed genes were involved in molecular function, cellular components and biological function. KEGG analysis indicated that genes were enriched in 13 pathways, of which four pathways were the most evident. These pathways were pathways in cancer, mitogen-activated protein kinase signaling, axon guidance and insulin signaling. Furthermore, QMSP demonstrated that methylation of mutL homolog, phosphatase and tensin homolog, runt-related transcription factor, Ras association family member, cadherin-1, O-6-methylguanine-DNA-methyltransferase and P16 genes had no obvious difference in 2D, 3D and Tis culture conditions. In conclusion, the culture method had no influence on DNA methylation in CRC cells.

Relationship between methylation status of Rap1 GTPase activating protein and colon cancer

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People's Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43% (50.35%), 21.37% (8.39%) and 17.43% (15.71%) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P 60 years old: 36.26% (62.62%) and 26.23% (76.42%); well-differentiated vs. moderately/poorly-differentiated: 21.98% (40.32%) vs. 42.74% (74.20%); TNM Ⅰ-Ⅱ vs Ⅲ-Ⅳ: 25.31% (48.27%) vs. 36.26% (75.55%); all P > 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer. Key words: Colon neoplasms; RAP1 GTPase activating protein; Methylation; Quantitative methylation specific polymerase chain reaction

MST1R methylation as a diagnostic biomarker in renal cell tumors

Abstract Introduction Renal cell tumors comprise both benign – oncocytoma – and malignant – clear cell renal cell carcinoma, papillary renal cell carcinoma, and chromophobe renal cell carcinoma – entities. Since the differential diagnosis among renal cell tumors is sometimes difficult on clinical, imaging and pathological grounds, and prognosis is quite dissimilar, epigenetic-based diagnostic biomarkers, specially promoter methylation, might be useful for accurate diagnosis and therapeutic planning. Materials and methods EpiTect Methyl II PCR Array was used to screen methylation status of 22 genes, involved in epithelial to mesenchymal transition. Quantitative real-time methylation specific polymerase chain reaction was performed for candidate gene validation, and methylation levels of renal cell tumors subtypes and normal kidney were determined and compared. Results MST1R promoter methylation level was significantly higher in clear cell renal cell carcinoma (median: 5367) compared to other renal cell tumors (median: papillary renal cell carcinoma – 1084, chromophobe renal cell carcinoma – 1023, oncocytoma – 1337) and normal kidney (median: 1125), allowing for accurate discrimination from other renal cell tumors with high sensitivity (>96.7%) and specificity (86.7%). Conclusion Quantitative MST1R promoter methylation may be useful as biomarker for accurate diagnosis of clear cell renal cell carcinoma in problematic cases.

Serum biomarkers for detection of head and neck squamous cell carcinoma.

Detection of hypermethylated circulating tumor DNA has the potential to be a minimally invasive, low cost, and reproducible method for cancer detection. We evaluated serum from 100 patients with known head and neck squamous cell carcinoma (HNSCC) and 50 healthy control patients for 3 previously described methylation targets, endothelin receptor type B (EDNRB), cyclin-dependent kinase inhibitor 2A (CDKN2A or p16), and deleted in colorectal carcinoma (DCC), using quantitative methylation specific polymerase chain reaction (qMSPCR). EDNRB hypermethylation was identified in the serum of 10% of the patients with HNSCC but in none of the control patients. DCC hypermethylation was detected in 2 serum samples from patients with cancer that also amplified EDNRB and one of these samples also had p16 hypermethylation. EDNRB hypermethylation was statistically significant by Fisher's exact test (p = .03) when comparing HNSCC to controls. Serum EDNRB hypermethylation is a highly specific but not sensitive serum biomarker for HNSCC.

Abstract 2850: Biomarkers for detection of exfoliated tumor cells in the peritoneal cavity in rectal cancer

Abstract During tumor growth, and possibly by manipulation during surgery, cells from rectal tumors may be shed into the peritoneal cavity. Such cells may contribute to development of metastatic disease, in the form of peritoneal carcinomatosis. Detection of cancer cells in the peritoneal cavity at the time of primary surgery might therefore be of value for prediction of disease recurrence with subsequent prognostic implications. We previously investigated the incidence of exfoliated cancer cells in peritoneal lavage fluid after resection of locally advanced rectal cancer (LARC). DNA from cells obtained by peritoneal lavage was analyzed with respect to mutations in hotspots of the K-RAS gene. K-RAS mutations were present in 30% of the primary tumors, and were detected in 8% of the lavage fluids examined. The presence of cells with K-RAS mutations in lavage fluid was associated with poor survival, suggesting that a molecular approach for detection of exfoliated tumor cells may be feasible, but K-RAS, although highly specific for detection of tumor cells, is insufficient as the only biomarker. Furthermore, since lavage fluid was collected only at the end of the surgical procedure, the relevance of spontaneous versus iatrogenic tumor cell exfoliation could not be determined. Based on these encouraging results, a prospective study was initiated, in which lavage is performed before and after surgical removal of the primary tumor to provide data regarding the cause of tumor cell exfoliation and their respective contributions to patient outcome. To increase detection sensitivity, a set of highly specific DNA methylation-based biomarkers is explored. In a pilot experiment, a panel of 12 biomarkers has been evaluated in primary tumors from 17 LARC patients using quantitative methylation specific polymerase chain reaction. Methylation of six genes, CNRIP1, MAL, SPG20, FBN1, SNCA and CDO1 was present at high frequencies in this cohort (in 55-88 % of the samples analyzed), and this biomarker panel has been selected for further comparative analysis in lavage samples. The presence of K-RAS, B-RAF and PIK3CA mutations is analyzed using denaturant capillary electrophoresis of PCR-amplified genes. Results from these genetic analyses of the primary tumors and lavage samples of 50 LARC patients will be presented. Citation Format: Annette T. Kristensen, Arne Solbakken, Stein G. Larsen, Karl-Erik Giercksky, Eivind Hovig, Ragnhild Lothe, Guro E. Lind, Marianne G. Guren, Kjersti Flatmark. Biomarkers for detection of exfoliated tumor cells in the peritoneal cavity in rectal cancer. [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 2850. doi:10.1158/1538-7445.AM2014-2850

Clinical Validation of an Epigenetic Assay to Predict Negative Histopathological Results in Repeat Prostate Biopsies

The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies. We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blindedpathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors. The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51). The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies.

Intraoperative peritoneal lavage: limitations of current evidence for clinical implementation.

Peritoneal recurrence after a curative resection of colorectal cancer is less common than lymphatic spread or hematogenous metastasis [1]. Moreover, peritoneal carcinomatosis often develops at a later stage of recurrent disease and is regarded as noncurable. Therefore, being able to predict the possibility of peritoneal recurrence at an early time after the original diagnosis and to prevent its occurrence would be a great advance in treating colorectal cancer [2]. Positive peritoneal lavage is a well-known risk factor for local/peritoneal recurrence in patients with colorectal cancer and various other types of cancers. Among gastrointestinal malignancies, the significance of peritoneal cytology has been extensively studied in gastric cancer. The existence of free cancer cells at intraoperative peritoneal lavage has been reported to be one of the most accurate prognostic factors for gastric cancer and is now classified as Stage IV [3, 4]. Besides gastric cancer, the examination of peritoneal free cancer cell is an important staging step in pancreatic, ovarian and endometrial cancer [5] Although positive peritoneal lavage is an established poor prognostic factor in colorectal cancer that has been proven in many studies, including several systemic reviews and meta-analyses [3, 6, 7], examining the peritoneal cytology is still not a standard procedure for staging colorectal cancer. To be a reliable clinical indicator, future studies have to overcome several issues involving peritoneal lavage. The first is the differences in the methods of lavage and free cell detection. Proper timing of lavage is controversial. Some studies performed lavage immediately after the opening of the abdominal wall and some so did after the tumor resection [7]. Most of these studies used a 0.9% NaCl solution as the lavage fluid, but the volume and the temperature of the solution were different from study to study. Most importantly, the methods for detecting free cancer cells varied and included conventional cytology, quantitative methylation specific polymerase chain reaction (PCR), immunological, immunobead quantitative PCR, radioimmunoassay or immunoperoxidase assay [8, 9, 10, 11, 12] methods. The staining technique was also different among the previous studies. These differences among the studies might result in a very wide range of positive peritoneal lavage ranging from 2.1% to 52%, and this wide range makes selecting a high-risk group for local/peritoneal recurrence based on peritoneal lavage difficult. Secondly, a well-designed comparative study is insufficient for recommending positive peritoneal cytology as a clinical indicator on which practical decisions can be based. A recent prospective study showed an improvement in peritoneal recurrence and overall survival in patients with positive lavage who underwent intraperitoneal chemotherapy with mitomycin C compared to those who did not [1, 13]. Because positive peritoneal lavage is associated with a worse prognosis, a potential benefit of peritoneal lavage may exist for the identification of patients to receive adjuvant treatment. Further studies are needed to examine whether adjuvant treatment can improve the oncologic results of patients with positive peritoneal lavage.

Aberrant TIG1 methylation associated with its decreased expression and clinicopathological significance in hepatocellular carcinoma

Recently, it has been reported that tazarotene-induced gene 1 (TIG1) methylation was frequently detected in a variety of human cancers. However, the relationship between the TIG1 methylation and the characteristics of hepatocellular carcinoma (HCC) remains unknown. The aim of present study was to observe the promoter methylation of TIG1 in HCC tissues and assess its prognostic significance for HCC. Real-time quantitative polymerase chain reaction and methylation-specific polymerase chain reaction were used, respectively, to examine the mRNA expression and methylation status of TIG1 in 91 pairs of HCC and adjacent noncancerous tissues. The mRNA expression level of TIG1 was significantly lower in HCC tissues than in adjacent noncancerous tissues. The rate of TIG1 promoter methylation was significantly higher in HCC tissues than in adjacent noncancerous tissues (P < 0.001). A strong correlation between downregulation and promoter methylation was found in these tumors (P < 0.001). More importantly, TIG1 methylation status was related to tumor size (P = 0.015), histological differentiation (P = 0.004), and tumor stage (P < 0.001). Kaplan-Meier survival analysis showed that TIG1 promoter hypermethylation was associated with a worse outcome in patients with HCC. Further, Cox multivariate analysis indicated that TIG1 methylation status was an independent prognostic factor for the overall survival rate of HCC patients. In conclusion, our data suggested that epigenetic silencing of TIG1 gene expression by promoter hypermethylation may play an important role in HCC.

MicroRNA-124 Regulates STAT3 Expression and Is Down-regulated in Colon Tissues of Pediatric Patients With Ulcerative Colitis

Altered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC). We used a library of 316 miRs to identify those that regulate phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time polymerase chain reaction analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2'-deoxycytidine (5-AZA). Methylation of the MIR124 promoter was measured by quantitative methylation-specific polymerase chain reaction. Levels of phosphorylated STAT3 and the genes it regulates (encoding vascular endothelial growth factor (VEGF), BCL2, BCLXL, and matrix metallopeptidase 9 [MMP9]) were increased in pediatric patients with UC compared with control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥ 75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was down-regulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 messenger RNA (mRNA). Levels of miR-124 were decreased, whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC compared with those with inactive disease. In addition, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Down-regulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-AZA up-regulated miR-124 and reduced levels of STAT3 mRNA. miR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and the pathogenesis of UC in children.

Clinical Utility of an Epigenetic Assay to Detect Occult Prostate Cancer in Histopathologically Negative Biopsies: Results of the MATLOC Study

Concern about possible false-negative prostate biopsy histopathology findings often leads to rebiopsy. A quantitative methylation specific polymerase chain reaction assay panel, including GSTP1, APC and RASSF1, could increase the sensitivity of detecting cancer over that of pathological review alone, leading to a high negative predictive value and a decrease in unnecessary repeat biopsies. The MATLOC study blindly tested archived prostate biopsy needle core tissue samples of 498 subjects from the United Kingdom and Belgium with histopathologically negative prostate biopsies, followed by positive (cases) or negative (controls) repeat biopsy within 30 months. Clinical performance of the epigenetic marker panel, emphasizing negative predictive value, was assessed and cross-validated. Multivariate logistic regression was used to evaluate all risk factors. The epigenetic assay performed on the first negative biopsies of this retrospective review cohort resulted in a negative predictive value of 90% (95% CI 87-93). In a multivariate model correcting for patient age, prostate specific antigen, digital rectal examination and first biopsy histopathological characteristics the epigenetic assay was a significant independent predictor of patient outcome (OR 3.17, 95% CI 1.81-5.53). A multiplex quantitative methylation specific polymerase chain reaction assay determining the methylation status of GSTP1, APC and RASSF1 was strongly associated with repeat biopsy outcome up to 30 months after initial negative biopsy in men with suspicion of prostate cancer. Adding this epigenetic assay could improve the prostate cancer diagnostic process and decrease unnecessary repeat biopsies.

IGFBP7 Promoter Methylation and Gene Expression Analysis in Prostate Cancer

IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer. We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens. IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples. To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

Role for p16 INK4a in progression of gastrointestinal stromal tumors of the stomach: alteration of p16 INK4a network members

Gastrointestinal stromal tumors feature a wide spectrum of biologic behavior, ranging from benign to extremely malignant. To determine the role of p16(INK4a) alteration in progression of gastrointestinal stromal tumors of the stomach, we have investigated protein expression and gene methylation in correlation with clinicopathologic factors and survival. In addition to immunohistochemical analysis of p16(INK4a) in a series of 95 cases, real-time quantitative methylation specific polymerase chain reaction for p16(INK4a) and immunostaining for cyclin D1, cyclin E, pRb, DP-1, E2F-1, and Ki-67 were also evaluated in randomly selected samples. The p16(INK4a) labeling indices ranged from 0% to 74% (median, 21%), demonstrating a significant inverse correlation with size (P = .046). On univariate (P = .003) and multivariate (P = .067) analyses, loss of p16(INK4a) expression increased the likelihood of a poor tumor-related survival. In addition, size (P = .036) and the mitotic index (P = .005) had independent prognostic influence. The p16(INK4a) methylation index, which ranged from 0% to 100% (median, 17%), was significantly higher in larger tumors (P < .001) and in high-risk category lesions (P= .001) and inversely correlated with protein expression. Hierarchical cluster analysis based on expression of p16(INK4a) network members identified 2 clusters in 27 randomly selected tumor samples, containing 11 and 16 tumors each. Former cluster samples demonstrated higher risk category (P = .022), higher p16(INK4a) methylation (P < .001), and more reduced pRb expression (P < .018). In addition, p16(INK4a) network members clustered into 2 groups: (1) showing down-regulated p16(INK4a) protein and up-regulating of both cyclin D1 and DP-1 and (2) down-regulated pRb and up-regulated E2F-1. We conclude that p16(INK4a) alteration has an important role in progression of gastrointestinal stromal tumors of the stomach. Furthermore, the study provides a possible link between regulation of p16(INK4a) network members and gastrointestinal stromal tumors.

Abstract 180: Potential utility of Reprimo methylation as a sensitizer to anticancer treatment in gastric cancer

Abstract Introduction: Reprimo is a highly glycosylated protein, inducing cell cycle G2 arrest. Reprimo promoter methylation has been reported as the mechanism regulating its expression in gastric cancer, but the biological roles remain unclear. The aim of this study is to examine Reprimo from a functional point of view. Methods: The methylation status of Reprimo promoter was analyzed using quantitative-methylation specific polymerase chain reaction (Q-MSP), and the effects of expression were examined in gastric cancer cell lines (AZ521 and SH10) with no endogenous expression of Reprimo using stable transfection and phenotype analysis (WST assay and apoptosis assay). Results: Reprimo methylation was detected in 6 of 8 gastric cancer cell lines; the silenced or reduced expression could be restored by treatment with demethylating agents such as 5-aza-2’-deoxycitidine and/or trichostatin A. On Q-MSP, the optimal cutoff value (0.42) determined by the receiver operating characteristic (ROC) curve could significantly discriminate primary tumor (69%, 57/83) from the corresponding normal tissues (18%, 15/83, P&amp;lt;0.0001). AZ521 cells with enforced expression significantly enhanced sensitivity to 5-FU therapy, and augmented apoptosis without affecting cell cycle. On the other hand, SH10 cells without methylation exhibited the undetectable level of endogenous expression, and Reprimo expression was robustly induced under 5-fluorouracil (5-FU), one of the standard anticancer agents for gastric cancer. As a result, SH10 cells stably transfected with Reprimo exhibited similar level apoptosis with mock cells. Conclusion: Reprimo expression is inducible under 5-FU agent, that may lead to apoptosis in gastric cancer. However, its expression is frequently suppressed by promoter methylation. Thus, Reprimo methylation status is considered to have a great potential as a predictive biomarker to select patients who may be effective for 5-FU therapy in gastric cancer. 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 180.

CpG Hypermethylation of the UCHL1 Gene Promoter is Associated With Pathogenesis and Poor Prognosis in Renal Cell Carcinoma

Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels. We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth. We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma. To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.