BACKGROUND: Although patients with chronic pancreatitis (CP) have an increased risk of pancreatic cancer (PanCa), the timely detection of PanCa often is difficult, because the symptoms of CP and PanCa are very similar. Moreover, secondary inflammation may be identified in PanCa, further complicating diagnosis. To improve the survival of patients with PanCa, a reliable test to differentiate CP from PanCa is needed. In this article, the authors describe a methylation profile of cell-free plasma DNA that distinguished CP from PanCa with >90% accuracy. METHODS: Methylation in cell-free, plasma DNA was compared among 30 samples from patients with CP, 30 samples from patients with PanCa, and 30 samples from healthy controls (N) using a microarray-mediated methylation analysis of 56 fragments in each sample (MethDet56). Statistical analysis was done by using the Fisher exact test, a naive Bayes algorithm, and 25 rounds of 5-fold cross-validation. RESULTS: The MethDet56 methylation analysis technique identified 17 gene promoters as informative (8 for distinguishing N from CP and 14 for distinguishing CP from PanCa). It achieved 81.7% sensitivity and 78% specificity (P<.01) in the detection of CP (N vs CP) and 91.2% sensitivity and 90.8% specificity (P<.01) in the differential detection of PanCa (PanCa vs CP). CONCLUSIONS: The current data suggested that, among patients with pancreatic disease, the methylation profiles of inflammatory disease and cancer are different and open a new venue for the development of biomarkers for differential diagnosis. Further investigation of diagnostic biomarkers for pancreatic cancer based on methylation in cell-free, circulating DNA appears to be warranted.
An accurate biomarker for detection of ovarian cancer may reduce cancer-related mortality. Using a previously developed microarray-based technique, we evaluated differences in DNA methylation profiles in a panel of 56 genes using sections of serous papillary adenocarcinomas and uninvolved ovaries (n ؍ 30) from women in a high-risk group. Methylation profiles were also generated for circulating DNA from blood of patients (n ؍ 33) and healthy controls (n ؍ 33). Using the most differentially methylated genes for naïve Bayesian analysis, we identified ten of these profiles as potentially informative in tissues. Various combinations of these genes produced 69% sensitivity and 70% specificity for cancer detection as estimated under a stratified , fivefold cross-validation protocol. In plasma , five genes were identified as informative; their combination had 85% sensitivity and 61% specificity for cancer detection. These results suggest that differential methylation profiling in heterogeneous samples has the potential to identify components of a composite biomarker that may detect ovarian cancer in blood with significant accuracy. Despite its relatively low prevalence, 1 ovarian cancer is the most frequent cause of death from gynecological malignancies. At early stages, women are mostly asymptomatic or present with vague and non-specific symptoms, so early ovarian cancer is difficult to diagnose. Considering that patients diagnosed with stage I ovarian cancer have a 5-year survival rate over 90%, early detection of ovarian cancer may reduce cancer-related mortality.It has been suggested that a screening test for ovarian cancer should have a positive predictive value of 10% or more; such that 10 women would undergo exploratory surgery to diagnose one cancer.2 The low prevalence of ovarian cancer requires that a screening test has a sensitivity of at least 75% and a specificity of at least 99.6%. 2The screening test should also be simple, inexpensive, and produce only minimal discomfort for the patient. Such a test has yet to emerge.The most widely used procedure for ovarian cancer detection and monitoring is a blood-based test for cancer antigen 125 (CA125).3,4 Its specificity for early-stage disease is high (96% to 100%), but the sensitivity is low, 5 so the test must be combined with other diagnostic techniques. A two-line screening procedure has been suggested in which candidates with high CA125 undergo follow-up transvaginal ultrasonography. 6 Unfortunately, this combination still has only a limited sensitivity because of the low sensitivity of the initial CA125 test.7 Even when high-risk women are screened, the test does not provide considerable advantages 8 ; it does not detect tumors early enough to influence outcomes.9 As a result, low sensitivity and a high rate of false-negative results of the CA125 test reduce access to transvaginal ultrasonography; on the other hand, the low sensitivity of transvaginal ultrasonography for early cancer suggests that the effect on prognosis would be negligible. 9To impro...
Objective-Epithelial ovarian carcinoma (OvCa) is rarely detected early, and it is also difficult to determine whether an adnexal mass is benign or malignant. Previously, we noted differences in methylation patterns of cell-free plasma DNA (cfpDNA) in women without disease compared to patients with OvCa. In this work we investigated whether methylation patterns of cfpDNA can differentiate between benign and malignant tumors.Methods-Methylation patterns in cfpDNA were determined in three cohorts (30 samples each) using a microarray-based assay (MethDet 56). Principal component analysis, supervised clustering, linear discrimination analysis, and 25 rounds of 5-fold cross validation were used to determine informative genes and assess the sensitivity and specificity of differentiating between OvCa vs. healthy control (HC), benign ovarian disease (mostly serous cystadenoma, BOD) vs. HC, and OvCa vs. BOD samples.Results-Differential methylation of three promoters (RASSF1A, CALCA, and EP300) differentiated between OvCa vs. HC with a sensitivity of 90.0% and a specificity of 86.7%. Three different promoters (BRCA1, CALCA, and CDKN1C) were informative for differentiating between BOD vs. HC, with a sensitivity of 90.0% and a specificity of 76.7%. Finally, two promoters (RASSF1A and PGR-PROX) were informative for differentiating between OvCa vs. BOD, with a sensitivity of 80.0% and a specificity of 73.3%.
Colorectal cancer (CRC) screening rates are currently suboptimal. Blood-based screening could improve rates of earlier detection for CRC and adenomatous colorectal polyps. In this study, we evaluated the feasibility of plasma-based detection of early CRC and adenomatous polyps using array-mediated analysis methylation profiling of 56 genes implicated in carcinogenesis. Methylation of 56 genes in patients with stage I and II CRC (N=30) and those with adenomatous polyps (N=30) were compared to individuals who underwent colonoscopy and were found to have neither adenomatous changes nor CRC. Composite biomarkers were developed for adenomatous polyps and CRC, and their sensitivity and specificity was estimated using five-fold cross validation. Six promoters (CYCD2, HIC1, PAX 5, RASSF1A, RB1, and SRBC) were selected for the biomarker, which differentiated CRC patients and controls with 84% sensitivity and 68% specificity. Three promoters (HIC1, MDG1, and RASSF1A) were selected for the biomarker, which differentiated patients with adenomatous polyps and controls with sensitivity of 55% and specificity of 65%. Methylation profiling of plasma DNA can detect early CRC with significant accuracy and shows promise as a methodology to develop biomarkers for CRC screening.
Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/μl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes.
Background-There is growing interest for identification of new targets for biomarker development in multiple sclerosis (MS). The goal of this study was to compare the concentration and the methylation patterns of cell-free plasma DNA (cfpDNA) in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals.
Abnormal DNA methylation is well established for cancer cells , but a methylation-based diagnostic test is yet to be developed. One of the problems is insufficient accuracy of cancer detection in heterogeneous clinical specimens when only a single gene is analyzed. A new technique was developed to produce a multigene methylation signature in each sample, and its potential for selection of informative genes was tested using DNA from formalin-fixed , paraffin-embedded breast cancer tissues. Fifty-six promoters were analyzed in each of 138 clinical specimens by a microarray-based modification of the previously developed technique. Specific methylation signatures were identified for atypical ductal hyperplasia , ductal carcinoma in situ, and invasive ductal carcinoma. Informative promoters selected by Fisher's exact test were used for composite biomarker design using naïve Bayes algorithm. All informative promoters were unmethylated in disease compared with normal tissue. Cross-validation showed 72.4% sensitivity and 74.7% specificity for detection of ductal carcinoma in situ and invasive ductal carcinoma, and 87.5% sensitivity and 95% specificity for detection of atypical ductal hyperplasia. These results indicate that informative cancer-specific methylation signatures can be detected in heterogeneous tissue specimens , suggesting that a diagnostic assay can then be developed. (J Mol
Differential methylation profiling of plasma DNA can detect ductal adenocarcinoma of the pancreas with significant accuracy and should be explored further. While additional improvement of biomarkers is necessary, the blood-based biomarker may be already useful as a first-line detection tool.
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