Panel Of DNA Methylation Markers Research Articles

Evaluation of Host Gene Methylation as a Triage Test for HPV-Positive Women-A Cohort Study.

This study was designed to evaluate the performance of a host gene methylation marker panel (ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671) in the triage of human papillomavirus (HPV)-positive women, its possible impact in a cervical cancer screening program, and the possible influence of the variation of the rate of HPV16/18 in its performance. Cohort study in which consecutive women referred for colposcopy in an organized cervical cancer screening program had repeated HPV testing, colposcopy, and biopsies. The women that remained HPV positive at the time of colposcopy were tested with the panel of DNA methylation markers. The performance of the test was evaluated and compared to standard practice. The study test had a sensitivity and specificity for cervical intraepithelial neoplasia (CIN) 2+ of 60.8% (49.1-71.6%) and 88.4% (83.2-92.5%), respectively. For CIN3+, it was of 78.0% (64.0-88.5%) and 86.0% (80.8-90.2%), respectively.The rate and level of methylation positively correlated with the severity of disease. The use of methylation reduces the referral for colposcopy to 25.5%, while detecting 78.0% of the CIN3+ cases. Referral of all HPV16/18-positive cases and triage of the other high-risk HPV-positive cases with methylation, detects 90.0% of the cases of CIN3+, while reducing the number of referrals to 43.2%.The variation in the rate of HPV16/18 does not relevantly affect the performance of the methylation panel. The studied methylation panel has a high sensitivity and specificity for CIN3+ and reduces the rate of referrals for colposcopy, without relevant variation according to the rate of HPV16/18.

DNA methylation biomarkers in colorectal cancer: Clinical applications for precision medicine.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide that is attributed to gradual long-term accumulation of both genetic and epigenetic changes. To reduce the mortality rate of CRC and to improve treatment efficacy, it will be important to develop accurate noninvasive diagnostic tests for screening, acute and personalized diagnosis. Epigenetic changes such as DNA methylation play an important role in the development and progression of CRC. Over the last decade, a panel of DNA methylation markers has been reported showing a high accuracy and reproducibility in various semi-invasive or noninvasive biosamples. Research to obtain comprehensive panels of markers allowing a highly sensitive and differentiating diagnosis of CRC is ongoing. Moreover, the epigenetic alterations for cancer therapy, as a precision medicine strategy will increase their therapeutic potential over time. Here, we discuss the current state of DNA methylation-based biomarkers and their impact on CRC diagnosis. We emphasize the need to further identify and stratify methylation-biomarkers and to develop robust and effective detection methods that are applicable for a routine clinical setting of CRC diagnostics particularly at the early stage of the disease.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer.

3545 Background: Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). Currently available assays can identify CMS1 and CMS4 cases well, while a dedicated test to distinguish CMS2 and 3 is lacking. This study aimed to identify a panel of methylation markers to distinguish between CMS2 and 3 in patients with CRC. Methods: Fresh-frozen tumor tissue of 239 patients with stage I-III CRC was included. CMS classification was performed on RNA-seq data using the single-sample-prediction parameter from the “CMSclassifier” package. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group-regularised logistic ridge-regression with post-hoc group-weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3 based on 15, 10 or 5 markers. Data from TCGAwas used for validation. Results: Overall methylation profiles differed between CMS2 and CMS3. Group-regularisation of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only 5 markers, sensitivity, specificity, and accuracy were > 90%. Validation showed comparable performances. Conclusions: Our highly sensitive and specific methylation marker panel can be used to distinguish CMS2 and 3. This enables development of a qPCR DNA methylation assay in patients with CRC to provide a specific and non-invasive classification tool.

Intrahepatic cholangiocarcinomas with IDH1/2 mutation-associated hypermethylation at selective genes and their clinicopathological features

Intrahepatic cholangiocarcinoma (ICC) is a rare but fatal tumor. The isocitrate dehydrogenase 1 and 2 (IDH1/2) genes are known to be mutated in ICC. IDH1/2 mutations tend to be accompanied by enhanced hypermethylation at a subset of genomic loci. We sought to clarify the clinicopathological features, including prognostic value, of ICCs with IDH1/2 mutation-associated hypermethylation at a subset of genes. The mutation status of IDH1/2 and methylation status of 30 gene CpG island loci were analyzed in 172 cases of ICC using pyrosequencing and the MethyLight assay, respectively. The mutation status of IDH1/2 was correlated with clinicopathological features and the DNA methylation status at 30 gene loci. Then, the clinicopathological characteristics were analyzed regarding three-tiered methylation statuses in genes showing IDH1/2 mutation-associated methylation. IDH1/2 mutations were found in 9.3% of ICCs, and IDH1/2-mutated tumors were associated with the histological subtype, including the bile ductular type and small duct type, and poor differentiation. Eight DNA methylation markers showed associations with IDH1/2 mutations, and ICCs with > 5/8 methylated markers were associated with the bile ductular type or small duct type, absence of mucin production, absence of biliary intraepithelial neoplasia, and presence of chronic liver disease. > 5/8 methylated markers were an independent prognostic marker associated with better survival in both cancer-specific survival and recurrence-free survival. In summary, by analyzing the association between IDH1/2 mutations and DNA methylation in individual genes, we developed a panel of DNA methylation markers that were significantly associated with IDH1/2 mutations and were able to identify a subset of ICC with better clinical outcomes.

Genome-wide analysis of DNA methylation identifies two CpG sites for the early screening of colorectal cancer.

Aim: To identify a panel of DNA methylation markers for the early diagnosis of colorectal cancer (CRC). Materials & methods: Using public omics data and our pyrosequencing data, we developed and validated a global methylation model and a CpG-methylation-based model for CRC screening. Results: Both of the models yielded high sensitivity and specificity for distinguishing CRC and its precursors (colorectal adenoma and colorectal laterally spreading tumor) from normal controls in eight independent datasets and our newly collected samples. More importantly, the two-CpG-based model showed high specificity in excluding inflammatory bowel diseases and other 13 cancer types. Conclusion: A diagnostic model based on two CpGs (cg09239744 and cg12587766) may be a powerful tool for CRC screening.

A panel of DNA methylation markers for the detection of prostate cancer from FV and DRE urine DNA

BackgroundEarly screening for prostate cancer (PCA) remains controversial because of overdiagnosis and overtreatment of clinically insignificant cancers. Even though a number of diagnostic tests have been developed to improve on PSA testing, there remains a need for a more informative non-invasive test for PCA. The objective of this study is to identify a panel of DNA methylation markers suitable for a non-invasive diagnostic test from urine DNA collected following a digital rectal exam (DRE) and/or from first morning void (FV). A secondary objective is to determine if the cumulative methylation is indicative of biopsy findings.MethodsDRE and FV urine samples were prospectively collected from 94 patients and analyzed using 24 methylation-specific quantitative PCR assays derived from 19 CpG islands. The methylation of individual markers and various combinations of markers was compared to biopsy results. A methylation threshold for cancer classification was determined using a target specificity of 70%. The average methylation and the number of positive markers were also compared to the result of the biopsy, and the area under the receiver operating characteristic curves (AUCs) were calculated.ResultsMethylation of all 19 markers was detected in FV and DRE DNAs. Combining the methylation of two or more markers improved on individual marker results. Using 6of19 methylated markers as the threshold for cancer classification yielded a specificity of 71% (95% CI, 0.57–0.86) from both DRE and FV and a sensitivity of 89% (95% CI, 0.79–0.97) from DRE and 94% (95% CI, 0.84–1.0) from FV. The negative predictive value at the 6of19 threshold was ≥ 90 for both DNA types.ConclusionsPCA-specific methylation was detected in both FV and DRE DNA. There was no significant difference in diagnostic accuracy at the 6of19 threshold between DRE and FV urine DNA. The results support the development of a non-invasive diagnostic test to reduce unnecessary biopsies in men with elevated PSA. The test can also provide patients with personalized recommendations based on their own methylation profile.

Abstract 4341: Employing the epigenetic field effect to detect prostate cancer in biopsy-negative patients

Abstract Purpose: Prostate cancer (PCa) is typically multifocal supporting the principle that molecular alterations can occur in histologically normal tissues in patients that have cancer. We have recently demonstrated using DNA methylation microarrays that epigenetic differences define a widespread field defect in the normal prostate tissue from men with cancer. The purpose of this study is to determine whether a novel panel of DNA methylation markers can predict the presence of PCa using fresh and paraffin-embedded histologically normal biopsy cores from multiple medical sites. Materials and Methods: Encode HG18 DNA methylation arrays, containing 385,000 probes tiling the biologically significant pilot regions of the human genome, were used to identify methylation alterations between NTA and TA tissues. Bisulfite Pyrosequencing confirmed alterations at numerous loci. Methylation was assessed using quantitative pyrosequencing in a training set consisting of 65 non-tumor associated (NTA) and tumor associated (TA) prostate tissues. A multiplex model was generated using multivariate logistic regression and externally validated in a blinded fashion using a set of 47 NTA and TA biopsy specimens from an external institution. Results: The methylation field defect was not significantly different between adjacent and distant tissues from tumor foci indicating this was not a peritumor effect. DNA methylation was analyzed in a subset of significant loci in NTA and TA tissues. Robust methylation differences were observed for all genes at all CpGs assayed (p<0.0001) in the 47 fresh-frozen specimens. Regression models incorporating individual genes (EVX1, CAV1, and FGF1) and a gene panel (EVX1 and FGF1) discriminated between NTA and TA tissues in the original training set (AUC 0.796-0.898, p<0.001). Upon external validation, uniplex models incorporating EVX1, CAV1, or FGF1 discriminated between TA and NTA biopsy-negative specimens with an AUC of 0.702, 0.696, and 0.658, respectively (p<0.05). Furthermore, a multiplex panel (EVX1 and FGF1) identified PCa patients with an AUC of 0.774 (p = 0.001) and had a negative predictive value of 0.909. Further results will be reported from an ongoing multicenter trial involving 160 FFPE specimens. Conclusions: A widespread epigenetic field defect can be utilized to detect the existence of PCa in patients with histologically negative biopsies. This assay is unique in that it detects alterations in non-tumor cells at distance from tumor foci. With further validation, this marker panel (EVX1 and FGF1) has the potential to decrease the need for repeated prostate biopsies, a costly procedure associated with pain and complications. Citation Format: Bing Yang, Johnathon McCormick, Nathan Damaschke, Anastasia Montgomery, Glen Leverson, Wei Huang, Daniel W. Lin, David F. Jarrard. Employing the epigenetic field effect to detect prostate cancer in biopsy-negative patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4341.

A panel of DNA methylation markers reveals extensive methylation in histologically benign prostate biopsy cores from cancer patients.

BackgroundMen with a negative first prostate biopsy will undergo one or more additional biopsies if they remain at high suspicion of prostate cancer. To date, there are no diagnostic tests capable of identifying patients at risk for a positive diagnosis with the predictive power needed to eliminate unnecessary repeat biopsies. Efforts to develop clinical tests using the epigenetic signature of cores recovered from first biopsies have been limited to a few markers and lack the sensitivity and specificity needed for widespread clinical adoption.MethodsWe developed methylation-specific quantitative polymerase chain reaction assays for a panel of 24 markers that are preferentially methylated in prostate cancer. We modified the bisulfite conversion conditions to allow the integration of the methylation information from multiple markers. We determined the methylation status of the 24 markers in 213 prostate biopsy cores from 104 patients, 37 prostate cancer patients and 67 controls. We performed logistic regression on combinations of markers as well as the entire panel of 24 markers to identify the best candidates for a diagnostic test.ResultsThe marker panel differentiated between cancer cores and benign cores from non-cancer patients with 100% sensitivity and 97% specificity. Furthermore, the panel detected significant methylation in benign cores from prostate cancer patients that was not present in controls. Using methylation of 5 out of 24 to define a cancer case, the analysis of a single benign biopsy core identified 62% of prostate cancer patients undergoing repeat biopsies. ROC curve analysis showed that markers commonly methylated in benign cores from cancer patients are the best candidates for a diagnostic test. The results suggest that 5 to 10 markers will be needed to achieve optimal predictive power.ConclusionsThis study shows that epigenetic field effects differ significantly between cancer patients and controls. Their detection in benign biopsy cores can form the basis of diagnostic tests to identify patients in need of repeat biopsies, reducing the cost of continued PCA screening by up to 40%. They could also be used to identify prostate cancer patients with low grade disease who are likely candidates for active surveillance or focal therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40364-014-0025-9) contains supplementary material, which is available to authorized users.

MP29-13 IMPROVING RENAL NEEDLE-BIOPSY ACCURRACY WITH RCC-SPECIFIC DNA METHYLATION MARKERS

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-13 IMPROVING RENAL NEEDLE-BIOPSY ACCURRACY WITH RCC-SPECIFIC DNA METHYLATION MARKERS Mehrdad Alemozaffar, Clayton Coolings, Sameer Chopra, Sumeet Syan, Brian Hu, Inderbir Gill, and Gangning Liang Mehrdad AlemozaffarMehrdad Alemozaffar More articles by this author , Clayton CoolingsClayton Coolings More articles by this author , Sameer ChopraSameer Chopra More articles by this author , Sumeet SyanSumeet Syan More articles by this author , Brian HuBrian Hu More articles by this author , Inderbir GillInderbir Gill More articles by this author , and Gangning LiangGangning Liang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.755AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The detection of small renal masses (SRMs) <4cm in size have risen given the widespread use of abdominal imaging. 20-30% of SRMs are benign and workup and management remains controversial with advocates for biopsy, surveillance, or surgical treatment. Thus, accurate characterization of SRMs is needed to determine treatment. Needle biopsy of SRMs have high false negative rates on standard microscopic examination (10–20%) since small amounts of tissue are sometimes obtained. We aimed to identify epigenetic markers to improve the diagnostic value of renal needle biopsies. METHODS Through an IRB approved protocol, needle biopsies were obtained ex-vivo following partial nephrectomy for 100 patients with SRMs. Each lesion underwent 3 pairs of biopsies: 2 from tumor regions and 1 from adjacent normal tissue; one was examined and other was used for DNA extraction. The methylation levels of these samples at select CpG dinucleotides throughout the genome were detected using the Infinium 450K array technology. To elucidate tissue-specific DNA methylation markers, these biological values were then compared to Infinium 450K data derived from 300 categorized renal samples from The Cancer Genome Atlas (TCGA). RESULTS Sixty SRMs have been examined to date. We isolated 9,432 candidate markers of differential DNA methylation with average beta value difference between two sample groups is >0.4 (Figure 1). Current efforts have been made to create an algorithm that allows us to predict the type of tissue based on methylation markers. To date, the algorithm has shown to successfully predict normal and cancerous tissue at a sensitivity rate of 91% and 90%, respectively. CONCLUSIONS Preliminary results suggest significant differences in DNA methylation levels exist at specific sites in the human genome among normal and aberrant kidney tissues. It's likely these biomarkers can be used to distinguish tissue types. We are using statistical approaches to identify a panel of DNA methylation markers to improve predicting phenotype and develop an economically efficient method for distinguishing between benign and cancer tissues. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e309-e310 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Mehrdad Alemozaffar More articles by this author Clayton Coolings More articles by this author Sameer Chopra More articles by this author Sumeet Syan More articles by this author Brian Hu More articles by this author Inderbir Gill More articles by this author Gangning Liang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DNA Methylation Markers Predict Outcome in Node-Positive, Estrogen Receptor-Positive Breast Cancer with Adjuvant Anthracycline-Based Chemotherapy

We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy. Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone. This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.

DNA methylation marker and prediction of outcome in node-positive, estrogen-receptor positive breast cancer patients receiving adjuvant anthracyclin-based chemotherapy

11046 Background: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multi-center study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor (ER)-positive, HER2-negative breast cancer patients who received adjuvant anthracyclin-based chemotherapy. Methods: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. Median follow-up was 82 months. Results: DNA methylation of PITX2 and 14 other genes was significantly correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A 4-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in further improvement of outcome prediction compared to PITX2 alone. Conclusions: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, 3 of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracyclin-treated primary breast cancer population. Our results demonstrate that a well- defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER2-negative breast cancer patients treated with anthracyclin-based chemotherapy. Considering the long-term toxicities of anthracyclines and new effective non-anthracycline combinations, patients with poor outcome may be candidates for non-anthracycline regimens. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Epigenomics Epigenomics

Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.