Methylation-sensitive Restriction Enzyme Research Articles

Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites.

We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5'-CG/5'-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (ΔA323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3' flanking position making it a CCA and CCC specific DNA methyltransferase.

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation.

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection.

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Non-small Cell Lung Cancer Epigenomes Exhibit Altered DNA Methylation in Smokers and Never-smokers

Epigenetic alterations are widespread in cancer and can complement genetic alterations to influence cancer progression and treatment outcome. To determine the potential contribution of DNAmethylation alterations to tumor phenotype in non-small cell lung cancer (NSCLC) in both smoker and never-smoker patients, we performed genome-wide profiling of DNA methylation in 17 primary NSCLC tumors and 10 matched normal lung samples using the complementary assays, methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation sensitive restriction enzyme sequencing (MRE-seq). We reported recurrent methylation changes in the promoters of several genes, many previously implicated in cancer, including FAM83A and SEPT9 (hypomethylation), as well as PCDH7, NKX2-1, and SOX17 (hypermethylation). Although many methylation changes between tumors and their paired normal samples were shared across patients, several were specific to a particular smoking status. For example, never-smokers displayed a greater proportion of hypomethylated differentially methylated regions (hypoDMRs) and a greater number of recurrently hypomethylated promoters, including those of ASPSCR1, TOP2A, DPP9, and USP39, all previously linked to cancer. Changes outside of promoters were also widespread and often recurrent, particularly methylation loss over repetitive elements, highly enriched for ERV1 subfamilies. Recurrent hypoDMRs were enriched for several transcription factor binding motifs, often for genes involved in signaling and cell proliferation. For example, 71% of recurrent promoter hypoDMRs contained a motif for NKX2-1. Finally, the majority of DMRs were located within an active chromatin state in tissues profiled using the Roadmap Epigenomics data, suggesting that methylation changes may contribute to altered regulatory programs through the adaptation of cell type-specific expression programs.

The relationship between obesity-related H19DMR methylation and H19 and IGF2 gene expression on offspring growth and body composition.

Imprinted genes are important for the offspring development. To assess the relationship between obesity-related H19DMR methylation and H19 and IGF2 gene expression and offspring growth and body composition. Thirty-nine overweight/obese and 25 normal weight pregnant women were selected from the "Araraquara Cohort Study" according to their pre-pregnancy BMI. Fetal growth and body composition and newborn growth were assessed, respectively, by ultrasound and anthropometry. The methylation of H19DMR in maternal blood, cord blood, maternal decidua and placental villi tissues was evaluated by methylation-sensitive restriction endonuclease qPCR, and H19 and IGF2 expression by relative real-time PCR quantification. Multiple linear regression models explored the associations of DNA methylation and gene expression with maternal, fetal, and newborn parameters. H19DMR was less methylated in maternal blood of the overweight/obese group. There were associations of H19DMR methylation in cord blood with centiles of fetal biparietal diameter (BPD) and abdominal subcutaneous fat thickness and newborn head circumference (HC); H19DMR methylation in maternal decidua with fetal occipitofrontal diameter (OFD), HC, and length; H19DMR methylation in placental villi with fetal OFD, HC and abdominal subcutaneous fat thickness and with newborn HC. H19 expression in maternal decidua was associated with fetal BPD and femur length centiles and in placental villi with fetal OFD and subcutaneous arm fat. IGF2 expression in maternal decidua was associated with fetal BPD and in placental villi with fetal OFD. To our knowledge, this is the first study to demonstrate associations of imprinted genes variations at the maternal-fetal interface of the placenta and in cord blood with fetal body composition, supporting the involvement of epigenetic mechanisms in offspring growth and body composition.

Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer

Methylation patterns in cell-free DNA (cfDNA) have emerged as a promising genomic feature for detecting the presence of cancer and determining its origin. The purpose of this study was to evaluate the diagnostic performance of methylation-sensitive restriction enzyme digestion followed by sequencing (MRE-Seq) using cfDNA, and to investigate the cancer signal origin (CSO) of the cancer using a deep neural network (DNN) analyses for liquid biopsy of colorectal and lung cancer. We developed a selective MRE-Seq method with DNN learning-based prediction model using demethylated-sequence-depth patterns from 63,266 CpG sites using SacII enzyme digestion. A total of 191 patients with stage I–IV cancers (95 lung cancers and 96 colorectal cancers) and 126 noncancer participants were enrolled in this study. Our study showed an area under the receiver operating characteristic curve (AUC) of 0.978 with a sensitivity of 78.1% for colorectal cancer, and an AUC of 0.956 with a sensitivity of 66.3% for lung cancer, both at a specificity of 99.2%. For colorectal cancer, sensitivities for stages I–IV ranged from 76.2 to 83.3% while for lung cancer, sensitivities for stages I–IV ranged from 44.4 to 78.9%, both again at a specificity of 99.2%. The CSO model's true-positive rates were 94.4% and 89.9% for colorectal and lung cancers, respectively. The MRE-Seq was found to be a useful method for detecting global hypomethylation patterns in liquid biopsy samples and accurately diagnosing colorectal and lung cancers, as well as determining CSO of the cancer using DNN analysis.Trial registration: This trial was registered at ClinicalTrials.gov (registration number: NCT 04253509) for lung cancer on 5 February 2020, https://clinicaltrials.gov/ct2/show/NCT04253509. Colorectal cancer samples were retrospectively registered at CRIS (Clinical Research Information Service, registration number: KCT0008037) on 23 December 2022, https://cris.nih.go.kr, https://who.init/ictrp. Healthy control samples were retrospectively registered.

Methylated SEPT9 assay-based liquid biopsy as a biomarker in molecular targeted agent-treated hepatocellular carcinoma.

The development of molecular targeted agents (MTAs) has changed the treatment strategy for hepatocellular carcinoma (HCC). However, currently, there are no established predictive biomarkers for the treatment efficacy of MTAs. Previously, we developed a novel liquid biopsy test for HCC screening using sensitive methylated DNA testing of septin 9 gene (SEPT9). Here, we hypothesized that SEPT9 could be used as a biomarker for MTA treatment efficacy. We enrolled 157 patients receiving sorafenib or lenvatinib as a first-line therapy and allocated 85 and 72 patients to the training and validation cohorts, respectively. For the methylation assay, DNA was treated with methylation-sensitive restriction enzymes, followed by multiplex droplet digital PCR. Various clinical parameters were compared with clinical outcomes. The multivariate analysis revealed Eastern Cooperative Oncology Group performance status (≥ 1; p = 0.048), alpha-fetoprotein (AFP) (≥ 400ng/mL; p < 0.001), and methylated-septin-9 (m-SEPT9) (≥ 205 copies/mL; p = 0.018) as significant predictors of poor overall survival (OS) in the training cohort. m-SEPT9 was identified as a predictor of poor OS in the validation cohort. We developed a predictive score, called the MTA score, consisting of these three significant OS parameters (two points were added for AFP and one point for each of the other predictors). Patients with MTA scores ≥ 2 showed a significantly poor prognosis compared to those with MTA scores ≤ 1 in both the training and validation cohorts. m-SEPT9 could be a potential predictive biomarker for survival in patients with HCC treated with MTAs.

Abstract 5434: Machine-learning-based epigenetic detection of early-stage lung cancers using the EpiCheck liquid biopsy platform

Abstract Alterations in DNA methylation are one of the earliest, most common signatures of cancer development, making them ideal biomarkers for early detection. Methylation profiling of plasma cell-free DNA (cfDNA) has significant potential to expand the use of liquid biopsies to cancer screening. The EpiCheck platform combines methylation-sensitive restriction endonuclease (MSRE) digestion with whole genome sequencing to comprehensively map genome-wide DNA methylation changes with high fidelity. In this proof-of-concept study, MSRE-NGS was used to interrogate a liquid biopsy atlas focused on early-stage lung cancers to i) identify informative NGS methylation biomarkers in plasma, and ii) develop a machine learning method to discriminate between cancer and high-risk, non-cancer controls. The EpiCheck lung cancer atlas was constructed using biospecimens from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks, and from a prospective multi-center study (NCT04968548). Plasma samples were collected from 93 high-risk primary lung cancer patients (62% stage I) and 90 high-risk individuals without cancer. Extracted DNA was digested with methylation-sensitive endonucleases and sequenced at an average depth of 600x. Methylation levels of ~6 million genomic loci were rank ordered using Student's t-test. Gene set enrichment analysis (GSEA) was performed on the top ranking 1000 differentially methylated loci. A logistic regression classifier with Lasso regularization was trained on 100,000 loci, and performance was examined by mean AUC using 5-fold cross-validation. A total of 23,130 loci exhibited significant differential methylation patterns between cancers and controls (p&amp;lt;0.01, FDR corrected). Of these, 20,159 and 2,971 lung cancer loci were hypermethylated and hypomethylated, respectively. Biological characterization using GSEA identified enrichments in transcriptional regulation and developmental control. In particular, loci were enriched for Polycomb Repressive Complex regulated genes, suggesting a possible connection to abnormal epigenetic regulation via histone modification in lung cancer. Construction of a machine learning logistic regression model based on the five training folds utilized 224 loci on average, and achieved a mean cross-validation AUC of 0.93 when distinguishing plasma cancer cases vs controls. Our findings demonstrate that the MSRE-NGS EpiCheck platform identified putative biomarkers within the plasma methylome for detecting early-stage lung cancer. A machine learning model trained on methylation targets performed with high accuracy in discriminating lung cancer patients from high-risk healthy individuals. Additional studies are required for defining the strength of the approach and for validating its use in non-invasive lung cancer screening. Citation Format: Dvir Netanely, Stephen Lam, Anna McGuire, Stephen Deppen, Eric Grogan, Fabien Maldonado, Michael Gieske, Joseph Seaman, Kimberly Rieger-Christ, Satish Kalanjeri, Luis Herrera, Nichole Tanner, Garrett B. Sherwood, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Revital Knirsh, Ofir Shliefer, Keren Manor, Radha Duttagupta, Aharona Shuali, Peter J. Mazzone, Gerard A. Silvestri, Catherine A. Schnabel, Danny Frumkin, Adam Wasserstrom. Machine-learning-based epigenetic detection of early-stage lung cancers using the EpiCheck liquid biopsy platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5434.

Abstract 3330: Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA

Abstract Lung cancer is the leading cause of cancer mortality, mainly due to diagnosis at advanced stages. Early detection reduces mortality and screening by CT is recommended for a high-risk population, however, uptake is low. Biomarkers for early-stage detection may enhance screening. Liquid biopsy and blood circulating tumor DNA (ctDNA) are established plasma surrogates of tumor tissue but their use in early detection remains challenging due to the high noise level of current techniques. We constructed a novel multiomic atlas through whole genome sequencing (WGS) to map all genetic and epigenetic changes associated with early-stage lung cancer as a tool to identify markers. Biospecimens from 48 early-stage lung cancer cases (58% stage I) and 29 cancer-free controls, all high-risk by USPSTF, were acquired from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks. Cases had tumor and normal lung tissue, whole blood (WB) and plasma samples. Controls had WB and plasma samples. EpiCheck sequencing (ECS) which combines methylation-sensitive restriction endonuclease digestion to detect differential methylation with WGS for multiomic analyses was performed on each sample. Extracted DNA was digested, underwent standard library preparation and sequenced at an average depth of 600x for cfDNA and 80x for tissue and WB. A subset also underwent WG bisulfite sequencing (BS) and standard WGS for comparison. Data analysis was performed using customized software. ECS outperformed BS. Mapping rate was 99.6%, 99.7% and 85.7% and unique mapping rate was 94.1%, 94.3% and 81.4% for WGS, ECS, and BS samples, respectively. Copy number integrity showed Pearson correlations of 0.9 for ECS and 0.67 for BS. Somatic mutation analysis in tissue identified a subset of cases with relatively high ctDNA shedding in plasma that were associated with larger tumors, older age and squamous cell carcinoma histology. This subset was further used to identify tumor derived plasma-based markers and assess fragmentation with high confidence. Shorter ctDNA fragments were observed in some, but not all cases. Additional analysis of WB and plasma identified host derived blood-based markers using methylation and copy number. This orthogonal approach enabled detection of small, low shedding tumors. A discovery panel of 87 markers showed 100% sensitivity and 97% specificity to discriminate cases vs controls. Only 11% of these markers are represented in the TCGA data. ECS generates high integrity sequencing data superior to BS, enabling genome-wide multiomic analyses (methylation, mutation, copy number, fragmentomics), with methylation signatures beyond the scope of TCGA. Results, which require validation, underscore the potential of the EpiCheck lung cancer atlas as a development platform of novel blood biomarkers for early-stage lung cancer detection. Citation Format: Revital Knirsh, Stephen Lam, Anna McGuire, Peter J. Mazzone, Stephen Deppen, Eric Grogan, Fabien Maldonado, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Dvir Netanely, Aharona Shuali, Catherine A. Schnabel, Adam Wasserstrom, Danny Frumkin. Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3330.

Abstract 3296: A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study

Abstract Objectives: Upper tract urothelial carcinoma (UTUC) is an uncommon, yet lethal tumor of the urinary tract. Diagnosis and preoperative risk stratification of these patients present distinct challenges given the limitations of current available diagnostic tools, including endoscopic biopsy and imaging. Herein, we explore the feasibility and clinical performance of a urine-based epigenetic assay in the diagnosis of patients with UTUC. Methods: Under an institutional review board-approved protocol, voided urine samples were prospectively collected using the Bladder CARE Urine Collection Kit (Pangea Laboratory LLC, CA, USA) from primary UTUC patients before any genitourinary manipulation between November 2019 and March 2022. All patients were confirmed to have UTUC in the final specimen. A 1:1 age/sex-matched group of cancer-free healthy donors (control group) was also included in the study. “Healthy” status was based on self-reporting and defined as no history of any type of tumor. Urine samples were analyzed with Bladder CARE, a urine-based test that measures the methylation level of 3 urothelial cancer-specific biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results were reported as Bladder CARE Index (BCI) score and categorized as “positive” (BCI &amp;gt; 5), “high risk” (2.5 &amp;lt; BCI ≤ 5) or “negative” (BCI ≤ 2.5). Association between BCI score and category, and clinical and pathological findings was assessed. Results: A total of 50 patients (40 males and 10 females) with a median age of 72 (64-79) years were included in the final analysis. Six patients received neoadjuvant chemotherapy. Surgical procedures included RNU (n=40), ureterectomy (n=7), and URS (n=3). All patients who underwent URS were confirmed to have UTUC through biopsy. Among UTUC patients, Bladder CARE test resulted positive in 47, high-risk in 1, and negative in 2 patients. Comparing Bladder CARE test results with tumor pathologic features demonstrated a statistically significant correlation between BCI values and tumor size (p = 0.009). UTUC patients had significantly higher BCI values compared to the controls (mean BCI 189.3 vs. 1.6, respectively; p &amp;lt; 0.0001). ROC curve showed an AUC of 0.9662 (95% CI = 0.9296-1). Sensitivity, specificity, PPV, and NPV of Bladder CARE to detect UTUC were 96%, 88%, 88.9%, and 95.7%, respectively. In comparison, the sensitivity of urine cytology in our cohort was 37%. Conclusions: In this prospective pilot study, the proposed urine-based epigenetic test (Bladder CARE) showed high sensitivity and negative predictive value in the diagnosis of patients with UTUC. In addition, the sensitivity of this test was exceedingly higher than the standard urine cytology. A larger sample size study to validate the accuracy of this test is the next step. Citation Format: Paolo Piatti, Alireza Ghoreifi, Sanam Seyedian, Yap Ching Chew, Benjamin Jara, Lucy Sanossian, Jeffrey Bhasin, Michael Basin, Taikun Yamada, Gerhard Fuchs, Sumeet Bhanvadia, Rene Sotelo, Andrew Hung, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, Hooman Djaladat. A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3296.

Abstract 3335: DNA methylation markers for the surveillance of non-muscle invasive bladder cancer: Results from a prospective pilot study

Abstract Objectives: Bladder cancer (BC) is a common urinary tract cancer with a variable clinical course. With recurrence as high as 70%, cystoscopy and urine cytology are routinely employed during follow-up of patients with a history of non-muscle invasive bladder cancer (NMIBC). Although multiple FDA approved urine-based tests for BC detection and surveillance exist, diagnostic accuracy of these urine-based assays is still suboptimal. Here, we evaluate the diagnostic value of a newly developed non-invasive DNA methylation-based test for surveillance of NMIBC. Methods: We included patients undergoing blue-light surveillance cystoscopy for NMIBC between February 2019 and September 2021. Urine samples were collected at each surveillance cystoscopy prior any genitourinary manipulation. Samples were analyzed with Bladder CARE, a urine-based test that measures the methylation level of 3 bladder cancer specific biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results are reported as Bladder CARE Index (BCI) score and categorized as “positive” (BCI &amp;gt; 5), “high risk” (2.5 &amp;lt; BCI ≤ 5) or “negative” (BCI ≤ 2.5). Association between BCI score and category, cytology and cystoscopy findings were assessed. Results: A total of 503 samples were collected from 159 patients (median age of 73, 77% male). 103 biopsies were performed during surveillance cystoscopies, of which 26 (25%) showed evidence of cancer recurrence.Bladder CARE was positive (22) or high-risk (4) in all the positive biopsies, while cytology was atypical only in 7 and highly suspicious in 2. Among 77 negative biopsies, Bladder CARE was positive in 27 collected from 20 patients, 8 of whom developed recurrence detected during subsequent follow ups. Cytology was atypical in 2 of these 8 recurrence patients. 3 patients with positive Bladder CARE results and normal cystoscopies developed upper tract urothelial carcinoma later. Bladder CARE test was able to predict the recurrence within a median of 7 months prior cystoscopy. The Receiver Operating Characteristic (ROC) curve using the BCI values demonstrated the sensitivity, specificity, positive predictive values, and negative predictive value of 93%, 65%, 73.5%, and 89.5%, respectively (Table 1). Conclusions: Urine cytology had low sensitivity and PPV for urothelial carcinoma in this cohort. Our findings demonstrated the necessity of more accurate urine biomarkers in the surveillance of NMIBC patients. Our preliminary results showed that Bladder CARE test has high sensitivity and can potentially predict future recurrence. Citation Format: Paolo Piatti, Sanam Ladi-Seyedian, Sidney Roberts, Farshad Sheybaee Moghadam, Alireza Ghoreifi, Jeffrey Bhasin, Benjamin Jara, Lucy Sanossian, Yap Ching Chew, Sumeet Bhanvadia, Hooman Djaladat, Anne Schuckman, Gangning Liang, Siamak Daneshmand. DNA methylation markers for the surveillance of non-muscle invasive bladder cancer: Results from a prospective pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3335.

MP63-10 THE DIAGNOSTIC VALUE OF URINARY CYTOLOGY VERSUS DNA METHYLATION URINE BIOMARKER TEST FOR SURVEILLANCE OF NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP63-10 THE DIAGNOSTIC VALUE OF URINARY CYTOLOGY VERSUS DNA METHYLATION URINE BIOMARKER TEST FOR SURVEILLANCE OF NON-MUSCLE INVASIVE BLADDER CANCER Seyedeh Sanam Ladi Seyedian, Margot Van Den Sigtenhorst Fijlstra, Sina Sobhani, Sidney Roberts, Natalie Liu, Andrew Udov, Farshad Sheybaee Moghadam, Alireza Ghoreifi, Paolo Piatti, Benjamin Jara, Lucy Sanossian, Hooman Djaladat, Anne Schuckman, Gangning Liang, and Siamak Daneshmand Seyedeh Sanam Ladi SeyedianSeyedeh Sanam Ladi Seyedian More articles by this author , Margot Van Den Sigtenhorst FijlstraMargot Van Den Sigtenhorst Fijlstra More articles by this author , Sina SobhaniSina Sobhani More articles by this author , Sidney RobertsSidney Roberts More articles by this author , Natalie LiuNatalie Liu More articles by this author , Andrew UdovAndrew Udov More articles by this author , Farshad Sheybaee MoghadamFarshad Sheybaee Moghadam More articles by this author , Alireza GhoreifiAlireza Ghoreifi More articles by this author , Paolo PiattiPaolo Piatti More articles by this author , Benjamin JaraBenjamin Jara More articles by this author , Lucy SanossianLucy Sanossian More articles by this author , Hooman DjaladatHooman Djaladat More articles by this author , Anne SchuckmanAnne Schuckman More articles by this author , Gangning LiangGangning Liang More articles by this author , and Siamak DaneshmandSiamak Daneshmand More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003321.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder cancer (BC) has a variable clinical course. With frequent recurrence, cystoscopy and urine cytology are routinely employed during follow-up of patients with a history of non-muscle invasive bladder cancer (NMIBC). Although there are multiple FDA approved urine-based tests for BC detection and surveillance, diagnostic accuracy of these urine-based assays is still suboptimal. Here, we compare the diagnostic value of urine cytology and a newly developed urine-based DNA methylation test for surveillance of NMIBC. METHODS: We included patients undergoing blue-light surveillance cystoscopy for NMIBC between February 2019 and September 2021. Urine samples were collected at each surveillance cystoscopy. Samples were analyzed with Bladder CARE, a urine-based assay that measures methylation levels of 3 bladder cancer specific biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results are reported as Bladder CARE Index (BCI) score and categorized as “positive” (BCI>5), “high risk” (2.5<BCI£5) or “negative” (BCI£2.5). Association between BCI score and category, cytology and cystoscopy findings were assessed. RESULTS: A total of 503 samples were collected from 159 patients (median age of 73, 77% male). 103 biopsies were performed during surveillance cystoscopies, of which 26 (25%) showed evidence of cancer recurrence. BCI was positive in all but four (high-risk) of the positive biopsies. While cytology was atypical in 7(27%) and highly suspicious in 2(8%) of the positive biopsies. Among 77 negative biopsies, BCI was positive in 27 samples from 20 patients, of whom 8 patients developed recurrence detected during subsequent follow ups. Cytology was only atypical in 2/8 patients. Three patients had normal cystoscopies and positive BCIs who developed upper tract urothelial carcinoma later. BCI was able to predict the recurrence within a median of 7 months prior. The Receiver Operating Characteristic (ROC) curve using the BCI values demonstrated the sensitivity, specificity, positive predictive values, and negative predictive value of 93%, 65%, 73.5%, and 89.5%, respectively. CONCLUSIONS: Urine cytology had low sensitivity and PPV for urothelial carcinoma in this cohort. Our findings demonstrated the necessity of more accurate urine biomarkers in the surveillance of NMIBC patients. Our preliminary results showed that Bladder CARE has high sensitivity and can potentially predict future recurrences. Source of Funding: Zymo Research Corp © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e875 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Seyedeh Sanam Ladi Seyedian More articles by this author Margot Van Den Sigtenhorst Fijlstra More articles by this author Sina Sobhani More articles by this author Sidney Roberts More articles by this author Natalie Liu More articles by this author Andrew Udov More articles by this author Farshad Sheybaee Moghadam More articles by this author Alireza Ghoreifi More articles by this author Paolo Piatti More articles by this author Benjamin Jara More articles by this author Lucy Sanossian More articles by this author Hooman Djaladat More articles by this author Anne Schuckman More articles by this author Gangning Liang More articles by this author Siamak Daneshmand More articles by this author Expand All Advertisement PDF downloadLoading ...

Nonrandom X Chromosome Inactivation Detection

X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X-linked disorders, and evaluating the pathogenicity of a genetic variant identified in an X-linked gene. The protocols in this article utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation-sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can be used to calculate the ratio of inactivation between the two alleles that ultimately reflects whether a female has a random or nonrandom pattern of X chromosome inactivation. © 2023 Wiley Periodicals LLC. Basic Protocol 1: X chromosome inactivation assay Basic Protocol 2: PCR amplification and labeling of digested and undigested DNA templates.

Cell-type dependent regulation of pluripotency and chromatin remodeling genes by hydralazine

BackgroundThe generation of induced pluripotent stem cells has opened the field of study for stem cell research, disease modeling and drug development. However, the epigenetic signatures present in somatic cells make cell reprogramming still an inefficient process. This epigenetic memory constitutes an obstacle in cellular reprogramming. Here, we report the effect of hydralazine (HYD) and valproic acid (VPA), two small molecules with proven epigenetic activity, on the expression of pluripotency genes in adult (aHF) and neonatal (nbHF) human fibroblasts.MethodsaHF and nbHF were treated with HYD and/or VPA, and viability and gene expression assays for OCT4, NANOG, c-MYC, KLF4, DNMT1, TET3, ARID1A and ARID2 by quantitative PCR were performed. aHF and nbHF were transfected with episomal plasmid bearing Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and exposed to HYD and VPA to determine the reprogramming efficiency. Methylation sensitive restriction enzyme (MSRE) qPCR assays were performed on OCT4 and NANOG promoter regions. Immunofluorescence assays were carried out for pluripotency genes on iPSC derived from aHF and nbHF.ResultsHYD upregulated the expression of OCT4 (2.5-fold) and NANOG (fourfold) genes but not c-Myc or KLF4 in aHF and had no significant effect on the expression of all these genes in nbHF. VPA upregulated the expression of NANOG (twofold) in aHF and c-MYC in nbHF, while it downregulated the expression of NANOG in nbHF. The combination of HYD and VPA canceled the OCT4 and NANOG overexpression induced by HYD in aHF, while it reinforced the effects of VPA on c-Myc expression in nbHF. The HYD-induced overexpression of OCT4 and NANOG in aHDF was not dependent on demethylation of gene promoters, and no changes in the reprogramming efficiency were observed in both cell populations despite the downregulation of epigenetic genes DNMT1, ARID1A, and ARID2 in nbHF.ConclusionsOur data provide evidence that HYD regulates the expression of OCT4 and NANOG pluripotency genes as well as ARID1A and ARID2 genes, two members of the SWI/SNF chromatin remodeling complex family, in normal human dermal fibroblasts.

Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes.

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

DNA Methylation and Prospects for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy for Triple-Negative and Luminal B Breast Cancer.

Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction.

Raw data of sequencing experiment contained in the tentative paper "Methylation-sensitive restriction enzyme - droplet digital PCR (MSRE-ddPCR) assay for the high-sensitive analysis of DNA methylation hotspots"

Raw data of sequencing experiment contained in the tentative paper "Methylation-sensitive restriction enzyme - droplet digital PCR (MSRE-ddPCR) assay for the high-sensitive analysis of DNA methylation hotspots"

Raw data of sequencing experiment contained in the tentative paper "Methylation-sensitive restriction enzyme - droplet digital PCR (MSRE-ddPCR) assay for the high-sensitive analysis of DNA methylation hotspots"

Raw data of sequencing experiment contained in the tentative paper "Methylation-sensitive restriction enzyme - droplet digital PCR (MSRE-ddPCR) assay for the high-sensitive analysis of DNA methylation hotspots"

A Urine-based DNA Methylation Marker Test to Detect Upper Tract Urothelial Carcinoma: A Prospective Cohort Study.

We explored the accuracy of a urine-based epigenetic test for detecting upper tract urothelial carcinoma. Under an Institutional Review Board-approved protocol, urine samples were prospectively collected from primary upper tract urothelial carcinoma patients before radical nephroureterectomy, ureterectomy, or ureteroscopy between December 2019 and March 2022. Samples were analyzed with Bladder CARE, a urine-based test that measures the methylation levels of 3 cancer biomarkers (TRNA-Cys, SIM2, and NKX1-1) and 2 internal control loci using methylation-sensitive restriction enzymes coupled with quantitative polymerase chain reaction. Results were reported as the Bladder CARE Index score and quantitatively categorized as positive (>5), high risk (2.5-5), or negative (<2.5). The findings were compared with those of 1:1 sex/age-matched cancer-free healthy individuals. Fifty patients (40 radical nephroureterectomy, 7 ureterectomy, and 3 ureteroscopy) with a median (IQR) age of 72 (64-79) years were included. Bladder CARE Index results were positive in 47, high risk in 1, and negative in 2 patients. A significant correlation was found between Bladder CARE Index values and tumor size. Urine cytology was available for 35 patients, of whom 22 (63%) results were false-negative. Upper tract urothelial carcinoma patients had significantly higher Bladder CARE Index values compared to the controls (mean 189.3 vs 1.6, P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of the Bladder CARE test for detecting upper tract urothelial carcinoma were 96%, 88%, 89%, and 96%, respectively.Conclusions:Bladder CARE is an accurate urine-based epigenetic test for the diagnosis of upper tract urothelial carcinoma, with much higher sensitivity than standard urine cytology.