Real-time Methylation-specific Polymerase Chain Research Articles

MST1R methylation as a diagnostic biomarker in renal cell tumors

IntroductionRenal 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 methodsEpiTect 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. ResultsMST1R 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%). ConclusionQuantitative MST1R promoter methylation may be useful as biomarker for accurate diagnosis of clear cell renal cell carcinoma in problematic cases.

Detecting DNA Methylation of the BCL2, CDKN2A and NID2 Genes in Urine Using a Nested Methylation Specific Polymerase Chain Reaction Assay to Predict Bladder Cancer

Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

DNA methylation status of RARβ2 and p16(INK4α) in peripheral blood and tumor tissue in patients with esophageal squamous cell carcinoma

To study the methylation status of retinoic acid receptor β2 (RARβ2) and p16(INK4α) genes in peripheral blood and tumor tissues and the perioperative dynamic changes of free RARβ2 and p16(INK4α) in the peripheral blood, and to investigate the relationship between RARβ2 and p16(INK4α) methylation in peripheral blood and clinicopathological characteristics of esophageal squamous cell carcinoma (ESCC) and their value in evaluating the completeness of surgical resection. Real-time methylation specific polymerase chain reaction (real-time MSP) technique was used to detect the methylation status of RARβ2 and p16(INK4α) in tumor tissue, adjacent normal tissue and peripheral blood perioperatively in 76 cases of ESCC. Sixty age-matched healthy volunteers were randomly selected as a control. RARβ2 and p16(INK4α) hypermethylation presented in both tumor tissue [72.4% (55/76) and 86.8% (66/76)] and peripheral blood [63.2% (48/76) and 71.1% (54/76)] in the ESCC patients, showing a good agreement between them. RARβ2 and p16(INK4α) hypermethylation was significantly related with pathological stage, lymph node metastasis, and invasion of nerves and vessels (P < 0.05). The DNA methylation rate in peripheral blood was increasing first and then decreasing in the preoperative, intraoperative and postoperative periods. Moreover, the RARβ2 methylation in peripheral blood was shown to be significantly associated with family history of cancer (P = 0.023). RARβ2 and p16(INK4α) methylation in the peripheral blood in ESCC patients may reflect the tumor-bearing status in the body, and may serve as a valuable marker in assessment of the degree of completeness of surgical resection in ESCC patients.

Abstract 4483: Early life exposures and promoter methylation in breast cancer: the Western New York Exposures and Breast Cancer (WEB) Study

Abstract Abstract Background: Exposures in the peri- and post-natal period (in utero, infancy, childhood, and adolescence) are associated with breast cancer risk, particularly premenopausal breast cancer. Epigenetic alterations in response to these exposures may play a critical role in early life, and then influence breast cancer risk in adult life. Using archived tumor blocks from incident breast cancer cases in a population-based the Western New York Exposures and Breast Cancer Study (WEB Study), we examined the association between early life exposures and promoter methylation of three tumor suppressor genes (p16, E-cadherin, and RARα2). Method: DNA samples isolated from 803 paraffin embedded tumor tissue were analyzed for hypermethylation status using real time methylation-specific polymerase chain reaction (MSP). Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) for associations of early life exposure with the likelihood of promoter methylation. Results: Among premenopausal breast tumors, compared to those with normal birth weight (2.6-3.9 kg), there was increased likelihood of E - cadherin gene hypermethylation in tumors from lower birth weight (≥ 2.5 kg) (OR = 2.79, 95% CI, 1.15-6.82). Height was positively associated with the likelihood of hypermethylation of RAR-α2 gene in premenopausal tumors (OR =3.34, 95% CI, 1.19-9.39). For p16, there was significant differences in the likelihood of promoter methylation of p16 in relation to not having been breast fed in premenopausal tumors (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. The likelihood of promoter methylation of the individual genes did not differ by maternal age at delivery, maternal height, age at menarche, age of first live birth, and weight at age 20 in either strata of menopause Conclusion: We found lower birth weight, not having been breast fed, and higher height were associated with promoter methylation of E - cadherin, p16 and RAR-α2 premenopausal breast tumors, respectively, suggesting that early life exposures may affect breast carcinogenesis among younger women through aberrant DNA methylation. Further studies of more genes and larger sample size may provide additional insight. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4483. doi:1538-7445.AM2012-4483

A New Method for FMR1 Gene Methylation Screening by Multiplex Methylation-Specific Real-Time Polymerase Chain Reaction

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation in men. It is caused by abnormalities in the FMR1 gene that are associated with CGG repeat expansion and the hypermethylation status of its promoter. Methylated alleles lead to transcriptional inhibition and consequent loss of Fragile X Mental Retardation Protein. Chemical modification of cytosine to uracil by bisulfite treatment has proved to be an attractive method for DNA methylation studies that precludes labor-intensive Southern blot analysis, which is the gold standard test for FXS. In this report, bisulfite-treated DNA samples were amplified using real-time multiplex methylation-specific polymerase chain reaction followed by melting curve analysis. Our results show that all control samples with known CGG repeat numbers and methylation statuses were correctly diagnosed. The samples from 43 male patients were also successfully diagnosed, which were in complete agreement with the results of Southern blotting. By such means, 39 patients were found to have an unmethylated allele; 3, a fully mutated allele; and 1, both methylated and unmethylated alleles, thus implying a diagnosis of mosaicism. In conclusion, we find our method to be convenient for screening a large number of male patients with FXS, because it is rapid and easy to perform, especially when there is a low quantity of DNA that must be sensitively and accurately assayed.

Intraoperative autologous blood recovery in prostate cancer surgery:in vivovalidation using a tumour marker

Intraoperative autologous blood recovery during radical retro pubic prostatectomy has the potential of contamination with tumour cells. Its safety is proved by similar survival rates between allogeneic and autologous transfusion to oncology patients without standardization. Silencing of the gene encoding pi class of gluthatione-S transferase is a specific and sensitive molecular marker for prostate cancer, because it is present in more than 90% of prostate tumours. Using such tumour marker, we aimed to demonstrate that viable tumour cells could be eliminated using leucodepletion filters followed by irradiation. Fifty patients with pi class of gluthatione-S transferase promoter hypermethylation in their primary prostate tumours were included in the analysis. Peripheral blood samples were collected during anaesthetic induction and recovered blood was collected throughout the surgery and then submitted to washing, leucoreduction and irradiation. Samples were analysed stepwise for the presence of promoter hypermethylation using real-time methylation-specific polymerase chain reaction. Positive hypermethylation was found in recovered blood (two samples), recovered and washed blood (three samples), and recovered washed and filtered blood (two samples). After filtration and irradiation of the recovered blood, this marker could not be detected in any of the cases analysed, suggesting the absence of viable tumour cells. Even though the risk of disseminating tumour cells in prostate cancer surgery by intraoperative autologous blood recovery is not yet fully established, no tumour-specific gene amplification was found after the association of blood filtration and irradiation, suggesting a significant reduction of such risk.

DNA hypermethylation and clinicopathological features in breast cancer: the Western New York Exposures and Breast Cancer (WEB) Study

Aberrant DNA hypermethylation of gene promoter regions has been increasingly recognized as a common molecular alteration in carcinogenesis. We evaluated the association between major clinicopathological features and hypermethylation of genes in tumors among 803 incidence breast cancer cases from a large population-based case-control study conducted in Western New York State. DNA samples were isolated from archive paraffin embedded tumor tissue and were analyzed for hypermethylation status of the E-cadherin, p16, and RAR-beta(2) genes using real time methylation-specific polymerase chain reaction. The frequencies of hypermethylation were 20.0% for E-cadherin, 25.9% for p16, and 27.5% for RAR-beta(2) genes. For postmenopausal women, hypermethylation of E-cadherin tended to be more likely in progesterone receptor (PR) negative than in PR-positive tumors (odds ratio (OR), 1.41; 95% confidence interval (CI), 0.91-2.18). Hypermethylation of p16 tended to be more frequent among estrogen receptor (ER) negative cases than ER-positive cases (OR, 1.51; 95% CI, 1.01-2.32). Hypermethylation of RAR-beta(2) gene was inversely associated with histological and nuclear grade of breast cancer.

QUANTITATIVE GSTP1 METHYLATION LEVELS CORRELATE WITH GLEASON GRADE AND TUMOR VOLUME IN PROSTATE NEEDLE BIOPSIES

Methylation of the GSTP1 gene promoter region is the most common epigenetic change in prostate cancer. The quantitative GSTP1 methylation assay, a recently developed methylation specific and polymerase chain reaction based technique, allows the accurate discrimination of benign prostate tissue and prostate cancer even in small, formalin fixed prostate needle biopsies. In this study we investigated whether GSTP1 methylation correlated with Gleason grade and tumor volume in prostate biopsies. We also compared quantitative GSTP1 methylation with the histological diagnosis in each biopsy. Formalin fixed, paraffin embedded prostate needle cores (25 mu) were used for the GSTP1 methylation study. After excluding 98 prostate needle cores (47%) with insufficiently extracted DNA 109 biopsies from 40 patients with prostate cancer of various Gleason grades and tumor volume, and benign prostate tissue were tested for GSTP1 methylation using quantitative fluorogenic real-time methylation specific polymerase chain reaction. A total of 13 benign and 96 cancer biopsy parts were included in this study. GSTP1 methylation was positive (GSTP1-to-ACTB ratio 5 or greater) in 83 of 96 cancer parts (86.5%). All 13 benign parts (100%) were negative for GSTP1 methylation with a methylation level of 0 in 12 and 1 in 1. The sensitivity and specificity of quantitative GSTP1 methylation assay were 86.5% and 100%, respectively. Quantitative GSTP1 methylation correlated with Gleason grade, demonstrating higher GSTP1 methylation in higher Gleason grade tumors (Spearman correlation coefficient 0.377, p < 0.0001). Likewise, GSTP1 methylation also correlated with the cancer percent per biopsy (Spearman correlation coefficient 0.567, p < 0.0001). A multiple linear regression model predicted the greater contribution of cancer percent than Gleason grade to quantitative GSTP1 methylation levels. We confirmed that the quantitative GSTP1 methylation assay reliably discriminates benign and malignant prostate tissues, thus, augmenting the routine histological evaluation of prostate needle biopsies. In addition, quantitative GSTP1 methylation also correlated with prostate cancer Gleason grade and cancer volume, suggesting that quantitative GSTP1 methylation may be of prognostic significance.

Quantitative Gstp1 Methylation Clearly Distinguishes Benign Prostatic Tissue And Limited Prostate Adenocarcinoma

Hypermethylation of the glutathione S-transferase gene (GSTP1) is the most common (greater than 90%) reported epigenetic alteration in prostate cancer. It occurs early in cancer progression and it is a promising marker for detecting organ confined disease. To evaluate its use as a diagnostic tool for cancer we used quantitative GSTP1 methylation to test for the presence of cancer in 45 prostate needle biopsy samples. Paraffin tissue samples from 45 patients with minute foci of intermediate grade prostatic adenocarcinoma or benign disease on needle biopsy were tested for GSTP1 hypermethylation using quantitative fluorogenic real-time methylation specific polymerase chain reaction. This assay was performed in blinded fashion without previous knowledge of the histopathological diagnosis. DNA from 29 of the 45 paraffin samples was amenable to polymerase chain reaction amplification. In these 29 samples GSTP1 methylation was detected in 11 of 15 cases of limited cancer and in 0 of 14 of benign disease (2-sided Fisher's exact test, p <0.0001). Thus, this assay had 73% sensitivity, 100% specificity, 100% positive and 78% negative predictive values. Quantitation of GSTP1 hypermethylation accurately detects the presence of cancer even in small, limited tissue samples. It represents a promising diagnostic marker that could be used as an adjunct to tissue biopsy as part of prostate cancer screening.