Real-time Quantitative Methylation-specific PCR Research Articles

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.

Cell-based breast cancer risk stratification based on DNA methylation in fine needle aspiration samples

1508 Background: Tumor suppressor gene (TSG) methylation is identified in nearly all breast cancers, but rarely in histologically normal breast tissue from wonen unaffected with breast cancer. Its occurrence in high risk preneoplasia and in benign breast tissue adjacent to breast cancer suggests that it may represent a high risk field change that could be exploited for cell-based breast cancer risk stratification. Methods: TSG methylation was measured by quantitative methylation-specific real time PCR in 53 breast tumor fine needle aspiration (FNA) biopsies, 84 cellular random periareolar FNAs (RP-FNA) ipsilateral or contralateral to these cancers, 36 cellular RP- FNAs from unaffected women at high risk for breast cancer by the Gail model, and 95 cellular RP-FNAs from unaffected women at lower risk by the Gail model. Results: The breast tumors showed a high frequency of TSG methylation: RASSF1A 80%, HIN-1 65%, Cyclin D2 60%, RAR-β2 53%, and APC 47%. In general, RP-FNA samples from cancer patients and Gail high risk patients showed a greater frequency of methylation than samples from Gail lower risk patients: RASSF1A 43% vs. 21%, P = 0.001, HIN-1 32% vs. 20%, P = 0.05; Cyclin D2 18% vs. 9%, P = 0.10; RAR-β2 21% vs. 18%, P = 0.68; and APC 25% vs. 16%, P = 0.17. Twelve of 215 RP-FNA samples (5%) showed very high levels of methylation (>10% methylation for two or more genes). Only two of these samples were from women classified as lower risk by the Gail model. Methylation frequencies were entirely independent of cell yields but the frequency of RASSF1A methylation increased with increasing Masood scores (P = 0.05). Methylation of RASSF1A in one breast was highly predictive of RASSF1A methylation in the opposite breast (P < 0.0001). Conclusions: TSG methylation appears to be a breast cancer risk-associated field change that can be quantified in RP-FNA samples. RASSF1A methylation occurs frequently in benign breast epithelium, provides reasonable discrimination between high and lower risk breasts (O.R. = 2.0), is related to cytological atypia, and may be an early marker of a methylator phenotype. Quantification of TSG methylation in RP-FNA samples may provide a valuable surrogate endpoint biomarker for Phase II prevention trials. No significant financial relationships to disclose.

Molecular Detection of Localized Prostate Cancer Using Quantitative Methylation-Specific PCR on Urinary Cells Obtained Following Prostate Massage

The diagnosis of localized prostate cancer is difficult due to a lack of cancer-specific biomarkers. Many patients require repeat prostate biopsies to diagnose the disease. We investigated whether aberrant promoter hypermethylation in prostatic fluid could reliably detect prostate cancer. Urine samples were collected after prostate massage from 95 patients with localized prostate cancer undergoing radical prostatectomy (63 pT(1), 31 pT(2), and 1 pT(3)) and from 38 control patients. Ten genes (GSTP1, RASSF1a, ECDH1, APC, DAPK, MGMT, p14, p16, RARbeta2, and TIMP3) were investigated using quantitative real-time methylation-specific PCR. Receiver operator curves were generated. The frequency of gene methylation ranged from 6.3% (p14) to 83.2% (GSTP1) in prostate cancer patients. At least one gene was hypermethylated in 93% of cancer patients. The specificity of methylation was 0.74. Methylation was significantly more frequent (P < 0.05) in cancer than control patients for all genes except p14 and p16. According to receiver operator curve analysis, the four-gene combination of GSTP1 (0.86), RASSF1a (0.85), RARbeta2 (0.80), and APC (0.74) best discriminated malignant from nonmalignant cases. The sensitivity and accuracy of this four-gene set were 86% and 89%, respectively. The presence of aberrant methylation in urinary cells obtained after prostate massage is significantly associated with prostate cancer. A panel of four genes could stratify patients into low and high risk of having prostate cancer and optimize the need for repeat prostatic biopsies.

Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue

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

Assessment of tumor suppressor gene methylation for breast cancer risk screening

1004 Background: Tumor suppressor gene (TSG) methylation is frequently detected in benign proliferative breast tissue suggesting that it occurs early in breast carcinogenesis. If it can be screen-detected and is associated with breast cancer risk it could be exploited for breast cancer prevention. Methods: Nipple duct lavage (NDL) samples, obtained from 150 women selected to represent a wide range of breast cancer risk, were evaluated by quantitative methylation-specific real time PCR. High risk breasts were defined as those contralateral to a breast cancer (N = 63) and those of women with a 5-year Gail risk ≥ twice the age- and race-matched general population risk (N = 64). The prevelence of TSG methylation and marked atypia was compared for high risk and lower risk breasts using Chi-square. Data for breasts ipsilateral to a breast cancer are shown for comparison, but not included in the calculations for the high risk category. Results: Samples with adequate cellularity were obtained for 219 breasts (76%). The proportion of healthy breasts with ≥ 1% of the gene copies methylated was 13% for Cyclin D2, 19% for APC, 19% for HIN-1, 16% for RASSF1A, and 9% for RAR-beta. RAR-beta provided the best risk discrimination as 15% of high risk breasts were methylated at a level that exceeded the 95th percentile of the lower risk breasts (0.9% of gene copies methylated, P = 0.05). For the table , methylation fractions for all five genes were summed and the threshold for classifying a breast as positive was set to the 95th percentile of the lower risk breasts (methylation sum = 25.0%). Both methylation and marked atypia provide some discrimination between high and lower risk breasts; the combination, however, provides the best discrimination (24% marker positive for high risk versus 9% for lower risk, P = 0.02). Conclusions: TSG methylation in NDL samples is a marker of breast cancer risk that is complementary to cytology. [Table: see text] [Table: see text]

Regularly methylated novel pro‐apoptotic genes associated with recurrence in transitional cell carcinoma of the bladder

Epigenetic silencing of tumor suppressor genes by promoter hypermethylation has been shown for a variety of genes in bladder cancer. Various p53 target genes have been investigated, but only few demonstrated promoter hypermethylation when semiquantitative detection methods were applied. To address to the question whether promoter methylation of novel p53 effector genes is a common event in transitional cell carcinoma of the bladder, we selected the p53 target genes apoptotic protein-activating factor (APAF-1), Caspase 8 (CASP-8), death-associated protein kinase, (DAPK-1) and insulin-like growth-factor-binding protein-3 (IGFBP-3), performing quantitative methylation-specific real-time PCR. The individual level of methylation (normalized index of methylation) was correlated with clinicopathological features as well as the biological behavior of the superficial and muscleinvasive tumors. Tissue was obtained from 110 tumor patients and 20 patients without urological malignancy. The median follow-up of the tumor patients was 52 months. Hypermethylation of the promoter region in tumor specimens was common for APAF-1 (100%), DAPK-1 (74%) and IGFBP-3 (66%), but not for CASP-8 (3.6%). It was seen less frequently and with undetectable or low methylation levels in the normal urothelium group. The APAF-1 methylation levels significantly correlated with tumor stage and tumor grade. The APAF-1 and IGFBP-3 methylation levels were able to separate tumors with higher recurrence risk from low-risk tumors in nonmuscleinvasive and muscleinvasive tumors. In multivariate analysis, APAF-1 and IGFBP-3 methylation levels were independent prognostic markers for recurrence in superficial bladder tumors. This study provides new insights into the role of promoter methylation of selected p53 target genes. The extent of promoter methylation of specific genes offers additional prognostical information and is associated with the outcome in patients with nonmuscleinvasive and muscleinvasive bladder cancer.

Molecular Profiling and Classification of Sporadic Renal Cell Carcinoma by Quantitative Methylation Analysis

Purpose: Preoperative histologic classification of solid renal masses remains limited with current technology. We determine the utility of molecular profiling based on quantitative methylation analysis for characterization of sporadic renal cell carcinoma. Experimental Design: Primary renal cell carcinomas representing three different histologic subtypes were obtained from a total of 38 patients who underwent radical nephrectomy for suspected malignant disease. Genomic DNA was isolated from tumors and was subjected to sodium bisulfite modification. The normalized index of methylation (NIM) for each sample was determined by quantitative real-time methylation-specific PCR at 17 different gene promoters. Hierarchical cluster analysis was performed by using an unsupervised neural network with binary tree topology. Results: The majority of gene promoters that were analyzed in this study demonstrated very low levels of methylation (NIM RASSF1A gene promoter, however, was methylated in 30 of 38 (79%) cases. The frequency of RASSF1A methylation in papillary, clear-cell, and oncocytoma subtypes was 100, 90, and 25%, respectively. The highest levels of RASSF1A methylation were observed in the papillary (mean NIM = 78.9) and clear-cell (mean NIM = 13.4) subtypes. The vast majority of oncocytomas were completely unmethylated, and none demonstrated >1% methylation (mean NIM = 0.11). Hierarchical cluster analysis based on quantitative methylation levels resulted in stratification of sporadic renal cell carcinomas into their discrete histologic subtypes. Conclusions: Classification of sporadic renal cell carcinomas into histologic subtypes can be accomplished via multigene quantitative methylation profiling. Validation of this approach and selection of appropriate methylation markers may ultimately lead to use of this technology in the preoperative assessment of suspicious renal masses.

DNA Methylation Alterations in the Pancreatic Juice of Patients with Suspected Pancreatic Disease

Molecular markers of pancreatic neoplasia could aid in the evaluation of visible pancreatic lesions and indicate neoplasia invisible to imaging. We evaluated methylation-specific PCR (MSP) assays that detect aberrantly methylated DNA for their use as markers of pancreatic neoplasia. Methylation analysis was done on pancreatic juice collected endoscopically or surgically from 155 individuals with suspected pancreatic disease: 56 patients had pancreatic ductal adenocarcinoma, 17 had intraductal papillary mucinous neoplasms, 26 had symptomatic chronic pancreatitis, 12 controls lacked evidence of pancreatic disease, and 44 were asymptomatic individuals at increased risk of developing familial pancreatic cancer undergoing screening for pancreatic neoplasia. Pancreatic juice DNA was analyzed for promoter methylation using conventional MSP assays for 17 genes. For six genes, pancreatic juice methylation was quantified using real-time quantitative MSP (QMSP; Cyclin D2, FOXE1, NPTX2, ppENK, p16, and TFPI2). Quantifying pancreatic juice methylation using QMSP with a cutoff of >1% methylated DNA could better predict pancreatic cancer than detecting methylation using conventional MSP. In the endoscopic group, 9 of 11 patients with pancreatic cancer, but none of 64 individuals without neoplasia had > or =1% methylation for two or more of the best five QMSP assays (82% sensitivity and 100% specificity; P < 0.0001). The prevalence of pancreatic juice methylation in patients with chronic pancreatitis was less than in patients with pancreatic cancer but higher than in controls and similar to high-risk individuals. The detection and quantification of aberrantly methylated DNA in pancreatic juice is a promising approach to the diagnosis of pancreatic cancer.

Detection of Minimal Residual Disease in Precursor B Lymphoblastic Leukemia (B-ALL) Patients by a Novel Epigenetic DNA Methylation Biomarker.

Background: Increasing evidence indicates that the minimal residual disease in childhood precursor B lymphoblastic leukemia (B-ALL) can predict the clinical outcome of the patients. With the combination of multicolor flow cytometry and B-cell receptor gene rearrangement or gene translocation analysis, the majority of minimal residual disease can be detected at the 10−5 level. However, these methods may suffer from high cost and complexity and result in false negatives due to antigen shift or lack of genetic markers. Aberrant DNA hypermethylation in CpG islands of tumor suppressor genes is a hallmark of many human malignancies including B-ALL. Since the DNA methyltransferase 1 (DNMT1) is a component of replisome at the DNA replication fork, the methylation in cytosine is precisely duplicated in each cell cycle. Thus the DNA methylation status is maintained and can be detected in the minimal residual leukemic cells if the original clone has DNA methylation markers.Methods: We developed a new method for detection of minimal residual disease in B-ALL patients using a specific DNA methylation marker in the promoter region of the tumor suppressor gene DLC-1 (deleted in liver cancer -1). The quantitative real-time methylation specific PCR (qMSP) was utilized as a primary detection method and it was validated via combined bisulfite restriction analysis (COBRA). The qMSP uses two specifically designed amplification primers which are complementary only for bisulfite-treated methylated DNA sequences and an additional fluorogenic probe specificlly hybridizing methylated target amplicons.Results: By using highly sensitive qMSP assay, we demonstrated that DNA methylation of the DLC-1 gene promoter occurred in 15 out of 20 (75 %) of B-ALL patient bone marrow aspirate specimens and 2 (NALM-6 and MN60) out of 3 B-ALL cell lines. DLC-1 methylation was detected in multiple sequential specimens (up to 6 time points) from 3 childhood B-ALL patients and is comparable with morphologic, flow cytometry and B-cell receptor gene rearrangement (in 5 specimens) assessment. In addition, DLC-1 methylation in different types of specimens collected at the same time point including frozen bone marrow aspirate, unstained bone marrow aspirate slides, fresh peripheral blood, and unstained peripheral blood smear showed complete consistency. Finally, the analytic sensitivity of qMSP was determined by a series of dilution of tumor cell DNA (MN60) in normal human genomic DNA. Ten ng of tumor DNA can be detected in 1 μg of normal DNA. The co-efficient of variation (CV) of the intra- and inter- measurements was about 0.5% and 1.5% by Ct values, respectively.Conclusion: We have developed a novel real-time methylation specific PCR method to detect minimal residual disease in B-ALL patients. The method is sensitive, quantitative, simple and fast, and has the potential to be used for routine clinical minimal residual disease detection in majority of B-ALL patients. The analytic sensitivity and specificity of this method are compatible with flow cytometric and molecular analysis. A study with larger number of B-ALL patient specimens using this novel method is currently underway.

Methylation Assay for the Diagnosis of Lung Cancer on Bronchial Aspirates: A Cohort Study

Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4a)), retinoic acid receptor beta2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16(INK4a), and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.

Detection of the placental epigenetic signature of the maspin gene in maternal plasma

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk

Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC). Clinical neoplastic progression risk factors, such as age and the length of the esophageal BE segment, have been identified. However, improved molecular biomarkers predicting increased progression risk are needed for improved risk assessment and stratification. Using real-time quantitative methylation-specific PCR, we screened 10 genes (HPP1, RUNX3, RIZ1, CRBP1, 3-OST-2, APC, TIMP3, p16, MGMT, p14) for promoter hypermethylation in 77 EAC, 93 BE, and 64 normal esophagus (NE) specimens. A subset of genes manifesting significant differences in methylation frequencies between BE and EAC was then analysed in 20 dysplastic specimens. All 10 genes except p14 were frequently methylated in EACs, with RUNX3, HPP1, CRBP1, RIZ1, and OST-2 representing novel methylation targets in EAC and/or BE. p16, RUNX3, and HPP1 displayed increasing methylation frequencies in BE vs EAC. Furthermore, these increases in methylation occurred early, at the interface between BE and low-grade dysplasia (LGD). To demonstrate the silencing effect of hypermethylation, we selected the EAC cells BIC1, in which the HPP1 promoter is natively methylated, and subjected them to 5-aza-2'-deoxycytidine (Aza-C) treatment. Real-time RT-PCR indicated increased HPP1 mRNA levels after 3 days of Aza-C treatment, as well as decreased levels of methylated HPP1 DNA. Hypermethylation of a subset of six genes (APC, TIMP3, CRBP1, p16, RUNX3, and HPP1) was then tested in a retrospective longitudinal study of 99 BE and nine LGD specimens obtained from 53 BE patients undergoing surveillance endoscopy. Only high-grade dysplasia (HGD) or EAC were defined as progression end points. Two patient groups were compared: eight progressors (P) and 45 nonprogressors (NP), using Cox proportional hazards models to determine the relative progression risks of age, BE segment length, and methylation events. Multivariate analyses revealed that only hypermethylation of p16 (odds ratio (OR) 1.74, 95% confidence interval (CI) 1.33-2.20), RUNX3 (OR 1.80, 95% CI 1.08-2.81), and HPP1 (OR 1.77, 95% CI 1.06-2.81) were independently associated with an increased risk of progression, whereas age, BE segment length, and hypermethylation of TIMP3, APC, or CRBP1 were not independent risk factors. In combined analyses, risk was detectable up to, but not earlier than, 2 years preceding neoplastic progression. Hypermethylation of p16, RUNX3, and HPP1 in BE or LGD may represent independent risk factors for the progression of BE to HGD or EAC. These findings have implications regarding risk stratification, early EAC detection, and the appropriate endoscopic surveillance interval for patients with BE.

Molecular Profiling and Classification of Sporadic Renal Cell Carcinoma by Quantitative Methylation Analysis

Preoperative histologic classification of solid renal masses remains limited with current technology. We determine the utility of molecular profiling based on quantitative methylation analysis for characterization of sporadic renal cell carcinoma. Primary renal cell carcinomas representing three different histologic subtypes were obtained from a total of 38 patients who underwent radical nephrectomy for suspected malignant disease. Genomic DNA was isolated from tumors and was subjected to sodium bisulfite modification. The normalized index of methylation (NIM) for each sample was determined by quantitative real-time methylation-specific PCR at 17 different gene promoters. Hierarchical cluster analysis was performed by using an unsupervised neural network with binary tree topology. The majority of gene promoters that were analyzed in this study demonstrated very low levels of methylation (NIM <1.0). The RASSF1A gene promoter, however, was methylated in 30 of 38 (79%) cases. The frequency of RASSF1A methylation in papillary, clear-cell, and oncocytoma subtypes was 100, 90, and 25%, respectively. The highest levels of RASSF1A methylation were observed in the papillary (mean NIM = 78.9) and clear-cell (mean NIM = 13.4) subtypes. The vast majority of oncocytomas were completely unmethylated, and none demonstrated >1% methylation (mean NIM = 0.11). Hierarchical cluster analysis based on quantitative methylation levels resulted in stratification of sporadic renal cell carcinomas into their discrete histologic subtypes. Classification of sporadic renal cell carcinomas into histologic subtypes can be accomplished via multigene quantitative methylation profiling. Validation of this approach and selection of appropriate methylation markers may ultimately lead to use of this technology in the preoperative assessment of suspicious renal masses.

Optimal use of a panel of methylation markers with GSTP1 hypermethylation in the diagnosis of prostate adenocarcinoma.

In this study, we tested the ability of a panel of hypermethylation markers to improve the sensitivity of histologic prostate cancer detection in sextant needle biopsies. We obtained fresh-frozen sextant biopsies from 72 excised prostates and directly compared blinded histologic review and quantitative real-time methylation-specific PCR for hypermethylation of four genes, Tazarotene-induced gene 1 (TIG1), adenomatous polyposis coli (APC), retinoic acid receptor beta2 (RARbeta2), and glutathione S-transferase pi (GSTP1) to detect the presence of prostate cancer. Results were compared with the final surgical pathological review of the resected prostates as the gold standard. Histologic review alone detected carcinoma with a sensitivity of 64% (39 of 61 cases) and 100% specificity. Quantitative real-time methylation-specific PCR for TIG1, APC, RARbeta2, and GSTP1 detected carcinoma with a sensitivity of 70%, 79%, 89%, and 75%, respectively, with 100% specificity for all of the genes. Using this panel of methylation markers in combination with histology resulted in the detection of 59 of 61 (97%) cases of prostate with 100% specificity, a 33% improvement over histology alone. The use of a panel of methylation markers as an adjunct to histologic review may substantially augment prostate cancer diagnosis from needle biopsies.

Early occurrence of RASSF1A hypermethylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis.

Follicular epithelial cell-derived thyroid tumors are common neoplasms comprised mainly of benign thyroid adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTCs). Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been reported recently in thyroid cancers. To additionally investigate the roles of these two epigenetic/genetic alterations in thyroid tumor progression, we examined their occurrences and relationship in both benign and malignant thyroid neoplasms. With real-time quantitative methylation-specific PCR, we found that 4 of 9 (44%) benign adenomas, 9 of 12 (75%) follicular thyroid cancers tumors, and 6 of 30 (20%) of PTC tumors harbored promoter methylation in > or = 25% of RASSF1A alleles. Additional quantitative analysis revealed RASSF1A methylation only in BRAF mutation-negative PTCs. A similar inverse correlation of RASSF1A methylation with BRAF mutation was seen in thyroid tumor cell lines. Our results, therefore, suggest that epigenetic inactivation of RASSF1A through aberrant methylation is an early step in thyroid tumorigenesis. Like the previously reported mutually exclusive relationship between BRAF mutation and other Ras pathway components such as RET/PTC rearrangement, a mutually exclusive relationship also exists between BRAF mutation and RASSF1A methylation in thyroid tumorigenesis.

Biallelic inactivation of the RIZ1 gene in human gastric cancer.

The distal short arm of chromosome 1 is commonly deleted in a variety of human neoplasms including gastrointestinal cancer. Genetic alterations of the retinoblastoma protein-interacting zing-finger gene (RIZ)1 including loss of heterozygosity (LOH) at 1p36, frameshift mutations, and promoter hypermethylation were reported previously in several cancers. In this study, we evaluated RIZ1 in 30 primary gastric cancers and found frameshift mutations in two cases (6.7%). Moreover, using real-time quantitative methylation-specific PCR, methylation of the RIZ1 promoter was detected in 11 (37%) cases. In all 11 cases with methylation, inactivation of the second allele occurred through frameshift mutation, LOH or promoter methylation. Our results suggest that RIZ1 is a specific target of inactivation in human gastric cancer.