qMSP Assays Research Articles

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.

Methylation of RAS association domain family protein 1A as a biomarker of lung cancer

Promoter hypermethylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for development of molecular biomarkers. This study aimed to determine the prevalence of RAS association domain family protein 1A (RASSF1A) promoter hypermethylation in bronchial aspirates of patients with suspected lung cancer and to test whether this type of methylation assay could be used as a diagnostic adjunct to conventional cytology. Two hundred three bronchial aspirates from patients with suspected lung cancer were analyzed for RASSF1A hypermethylation by using a sensitive quantitative methylation-specific polymerase chain reaction (QMSP). RASSF1A hypermethylation was found in 88% (35 of 40), 28% (31 of 111), and 100% (6 of 6) of bronchial aspirates collected from patients diagnosed with small cell lung cancer, nonsmall cell lung cancer, and combined small cell lung cancer, respectively. No hypermethylation was detected in patients diagnosed with nonneoplastic lung disease (0 of 46). Depending on histologic subtype, up to 82% of cases presenting with a negative histology showed a positive methylation assay. The QMSP analysis of RASSF1A hypermethylation enabled a highly specific distinction between patients diagnosed with lung cancer and those with nonneoplastic lung disease. These results suggested that a QMSP assay is a promising molecular tool for diagnosis of primary lung cancer.

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.

Quantitative Methylation-Specific Polymerase Chain Reaction Gene Patterns in Urine Sediment Distinguish Prostate Cancer Patients From Control Subjects

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Intraoperative molecular margin analysis in head and neck cancer.

Tumor-specific molecular alterations in surgical margins have been shown to predict risk of local recurrence. However, assays used for these analyses are time-consuming and therefore cannot be used in the intraoperative setting. To detect and quantify tumor-specific methylated promoter sequences in surgical margins in a time frame suitable for intraoperative use. A novel quantitative methylation-specific polymerase chain reaction (QMSP) protocol. A total of 13 patients with head and neck squamous cell carcinoma (HNSCC) were initially characterized for molecular alterations in their tumor at the time of biopsy. Six primary tumors were found to harbor promoter hypermethylation for p16 and O6-methylguanine-DNA-methyltransferase (MGMT) genes. Rapid QMSP was then used to identify promoter hypermethylation of these genes in the surgical margins. Results were compared with standard intraoperative histologic frozen section analysis and with conventional QMSP. Using our rapid QMSP assay, we found that 3 patients had methylation-positive margins. Tumor margins from 2 patients were methylated for p16 alone, and margins from 1 patient were methylated for p16 and MGMT simultaneously. Molecular margin analysis was completed in less than 5 hours, a time frame appropriate for selected major HNSCC resections that require combined primary tumor resection, cervical lymphadenectomy, and complex reconstruction. This technique was comparable in sensitivity to conventional QMSP. Rapid molecular margin analysis using QMSP is feasible and may be performed intraoperatively in selected patients with HNSCC that requires extensive resection.