Abstract Objectives: Bladder cancer (BC) stands as the 5th most prevalent cancer in the USA, with over 83,000 new cases diagnosed in 2023. The absence of non-invasive diagnostic tools for sensitive early BC detection and treatment response monitoring poses a significant challenge. This study evaluates the clinical potential of the Bladder CARE™ Assay for early BC detection and monitoring treatment response in BC patients. Methods: Under an institutional review board-approved protocol, voided urine samples were prospectively collected from USC patients with prior BC history under surveillance/anticancer treatment, prior genitourinary manipulation. Enrollment spanned February 2019 to September 2021. Samples underwent analysis using the Bladder CARE™ Assay, a DNA methylation test for quantitative BC and upper tract urothelial carcinoma (UTUC) detection from urine. Results were reported as Bladder CARE Index (BCI) and samples were categorized as “positive” (BCI > 5), “low-positive” (2.5 < BCI < 5), or “negative” (BCI < 2.5). Correlations between BCI value and categories, and clinicopathological findings were assessed. Results: A total of 110 previously diagnosed BC patients (median age: 74; 86% male) were enrolled in this study. Within 36 months post-TURBT, 24 patients (21.8%) showed evidence of recurrence. Bladder CARE™ Assay detected all recurrences, averagely 7.35 months earlier than cystoscopy. Of 55 patients (50%) undergoing anticancer therapies (45 BCG, 4 MMC, 2 GEM, 1 9UT, 1 GEM/DOCE, 1 MMC/BCG, and 1 BCG, MMC, and GEM), 7 (12.7%) recur (non-responders) and 11 (20%) did not show evidence of recurrence by 18 months post-TURBT (responders); 18-month post-operative data was unavailable for 37 patients (67.3%), which could not be classified in responders or non-responders. Bladder CARE™ detected 85.7% of non-responders, with BCI increasing prior to positive histology (avg. BCI: 86.1). In responders, BCI remained stable in negative/low-positive range post-TURBT (avg. BCI: 2.5). In addition, data from pre- and post-TURBT assessments were available for 20 of the 110 enrolled patients. The Bladder CARE™ Assay demonstrated a decrease in BCI post-TURBT in 19 of these 20 patients (95%). Conclusions: This prospective pilot study underscores the Bladder CARE™ Assay's capacity to pre-emptively detect BC months ahead of the gold standard. Furthermore, its quantitative nature offers prognostic insight, enabling non-invasive monitoring of patient response to anticancer treatments. A larger-scale study is the next step to validating these promising findings. Citation Format: Paolo Piatti, Sia Daneshmand, Sanam Ladi Seyedian, Saum Ghodossipour, Hamed Ahmadi, Suzanne Roberts, Alireza Ghoreifi, Michael Basin, Simin Hajian, Yap Ching Chew, Jeffrey Bhasin, Benjamin Jara, Lucy Sanossian, Hooman Djaladat, Anne Shuckman, Sumeet Bhavandia, Gangning Liang. DNA methylation biomarkers for early bladder cancer detection and treatment response monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB327.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP05)1 May 2024MP05-09 A NOVEL METHOD TO IDENTIFY AGGRESSIVE PROSTATE CANCER WITH DNA-METHYLATION SIGNATURES FROM PROSTATE NEEDLE BIOPSY Masatomo Kaneko, Yohei Sekino, Hongtao Li, Xinyi Zhou, Mingda Jin, Wei Guo, Xiaojing Yang, Jeffrey Bhasin, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E. Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, and Andre Luis Abreu Masatomo KanekoMasatomo Kaneko , Yohei SekinoYohei Sekino , Hongtao LiHongtao Li , Xinyi ZhouXinyi Zhou , Mingda JinMingda Jin , Wei GuoWei Guo , Xiaojing YangXiaojing Yang , Jeffrey BhasinJeffrey Bhasin , Atsuko FujiharaAtsuko Fujihara , Tsuyoshi IwataTsuyoshi Iwata , Lorenzo Storino RamacciottiLorenzo Storino Ramacciotti , Divyangi ParalkarDivyangi Paralkar , Giovanni E. CacciamaniGiovanni E. Cacciamani , Manju AronManju Aron , Osamu UkimuraOsamu Ukimura , Inderbir S. GillInderbir S. Gill , Gangning LiangGangning Liang , and Andre Luis AbreuAndre Luis Abreu View All Author Informationhttps://doi.org/10.1097/01.JU.0001008740.27639.cc.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To discover DNA-methylation signatures associated with clinically significant prostate cancer (CSPCa, grade group [GG] ≥2) on prostate biopsy (PBx). METHODS: Patients undergoing radical prostatectomy (RP) for prostate cancer (PCa) from April 2019 to July 2022 were prospectively recruited (IRB# HS-16-00404). Patients who underwent previous treatment for PCa were excluded. Two-core PBx fresh tissue was precisely sampled from the same location in the right and left lobes of the ex-vivo RP specimen (total 4-core per-patient). Then, one-core each was histopathologically (H&E) analyzed, and the other core was processed using Simplified Whole-panel Amplification Reaction Method (SWARM®) a robust targeted bisulfite sequencing approach. From 396,020 CpG sites (CpGs) related to PCa, 1254CpGs were selected for the analysis. The ex-vivo PBx histopathology was used as ground truth for paired-cores to identify methylation CpG sites (CpGs) strongly associated with CSPCa. The multi-class (benign vs GG1 vs CSPCa) discriminating performance was analyzed with a random forest. The samples were randomly separated at a 6:4 ratio into training and testing datasets. The area under the receiver operating characteristic curve (AUC) and diagnostic performance at Youden index on the testing dataset were reported. RESULTS: A total of 334 ex-vivo PBx cores (205 training vs 129 testing) acquired from 149 participants were included. The median age, PSA, and PSA density were 65 years, 6.7ng/mL, and 0.14ng/mL2, respectively. The index lesion on RP histology was GG1 (6.0%), 2 (52%), 3 (25%), 4 (4.0%), and 5 (12%). Benign tissue, GG1 and CSPCa were sampled in 230 (69%), 27 (8.1%) and 77 (23%) ex-vivo biopsy cores, respectively. Individual 44 CpGs as a feature in classifying PCa showed an average AUC of 0.80. The combined set of 44 CpGs achieved AUC 0.88 for PCa detection. Among them, the combination of top 12 CpGs showed AUC of 0.87, sensitivity of 78%, and specificity of 90% for CSPCa detection (Figure 1). CONCLUSIONS: Twelve DNA-methylation CpGs associated with CSPCa were successfully identified in PBx. This novel methodology presents the potential to distinguish patients with poor prognoses from PBx. Download PPT Source of Funding: None © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e46 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Masatomo Kaneko More articles by this author Yohei Sekino More articles by this author Hongtao Li More articles by this author Xinyi Zhou More articles by this author Mingda Jin More articles by this author Wei Guo More articles by this author Xiaojing Yang More articles by this author Jeffrey Bhasin More articles by this author Atsuko Fujihara More articles by this author Tsuyoshi Iwata More articles by this author Lorenzo Storino Ramacciotti More articles by this author Divyangi Paralkar More articles by this author Giovanni E. Cacciamani More articles by this author Manju Aron More articles by this author Osamu Ukimura More articles by this author Inderbir S. Gill More articles by this author Gangning Liang More articles by this author Andre Luis Abreu More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with high-multiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true predictive features and penalize confounding signals in predicting cancer diagnosis. Citation Format: Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia. Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3493.

PURPOSE:We explored the accuracy of a urine-based epigenetic test for detecting upper tract urothelial carcinoma. MATERIALS AND METHODS: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. RESULTS: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.

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 > 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.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.

Circulating cell-free DNA (cfDNA) has been reported to contain valuable genetic and epigenetic information for the diagnosis and prognosis of cancer. Studies have shown that the blood from cancer patients contained more tumor-derived cfDNA compared to the healthy controls. DNA methylation, the most well-studied epigenetic marker, has been validated as one of the key drivers in the development of many diseases including cancer. cfDNA methylation thus holds great potential to become a biomarker that will enable early detection of cancer. However, cfDNA is also a challenging input for library preparation as cfDNA is mostly highly fragmented, and the amount of cfDNA from blood plasma is comparatively low. Therefore, multiple library preparation protocols were evaluated for profiling DNA methylation patterns at single nucleotide resolution from ultra-low amount of blood plasma cfDNA. These methods included two bisulfite conversion-based protocols and an enzymatic based protocol. The bisulfite conversion reaction was optimized allowing a milder treatment for less nucleic acid damage. One of the bisulfite conversion based protocols also contained a further optimized adapter ligation step, allowing a simpler procedure. Circulating cfDNA were extracted from blood plasma of a healthy control, a non-small cell lung cancer patient, and an adenocarcinoma lung cancer patient. 5-ng cfDNA was used as input and the libraries were successfully prepared using all three methods. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling a mean coverage of about 10X per detected CpG, and over 90% of the CpG sites in human genome were detected. Unique alignment rate among all libraries were about or above 80%. The comprehensive coverage of the CpG sites across the entire human genome illustrated that the optimized bisulfite conversion reaction was compatible with fragmented cfDNA, proving the satisfactory efficacy in this simple and classic method for DNA methylation profiling of such a challenging sample type. This opens the door to apply the streamlined and simple Whole-Genome Bisulfite Sequencing (WGBS) protocol for cfDNA methylation profiling, which in turn shall facilitate further understanding in cfDNA epigenetic variations and advance the development of a cancer biomarker from the non-invasive, liquid biopsies. Citation Format: Caila Ruiz, Hanjun Kim, Zhenguo Zhang, Jeffrey Bhasin, Mingda Jin, Yi Xu, Xiaojing Yang. Applications of innovative whole-genome bisulfite sequencing (WGBS) for cancer biomarker discovery in plasma liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3762.

Background Bladder cancer (BC) is the 5th most common cancer in the USA. Non-muscle invasive bladder cancer represents about 70% of all cases and has generally a favorable outcome. However, recurrence rates as high as 60 to 70% and progression rates of 10 to 20% necessitate intensive surveillance with cystoscopy. The invasiveness and high cost of cystoscopy poses significant burden on BC patients as well as on the healthcare system. In this study we test the feasibility of a simple, sensitive, and non-invasive detection of BC using Bladder CARE test in urine samples. Results Urine from 136 healthy and 77 BC subjects was collected using the at-home Bladder CARE Urine Collection Kit and analyzed with Bladder CARE test. The test measures the methylation level of three BC-specific biomarkers and two internal controls using methylation-sensitive restriction enzymes coupled with qPCR. Bladder CARE showed an overall sensitivity of 93.5%, a specificity of 92.6%, and a PPV and NPV of 87.8% and 96.2%, respectively. Bladder CARE has an LOD as low as 0.046%, which equates to detecting 1 cancer cell for every 2,200 cells analyzed. We also provided evidence that bisulfite-free methods to assess DNA methylation, like Bladder CARE, are advantageous compared to conventional methods that rely on bisulfite conversion of the DNA. Conclusion Highly sensitive detection of BC in urine samples is possible using Bladder CARE. The low LOD of the test and the measurement of epigenetic biomarkers make Bladder CARE a good candidate for the early detection of BC and possibly for the routine screening and surveillance of BC patients. Bladder CARE and the at-home urine sample collection system have the potential to (1) reduce unnecessary invasive testing for BC (2) reduce the burden of surveillance on patients and on the healthcare system, (3) improve the detection of early stage BC, and (4) allow physicians to streamline the monitoring of patients.

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Abstract The information content of the human transcriptome is expanded by the presence of alternative mRNA isoforms produced by alternative transcription initiation, alternative splicing, and alternative polyadenylation. Alternative isoforms can possess distinct oncogenic and tumor suppressive functions by altering gene products and may therefore relate to aggressiveness of disease. A major challenge in prostate cancer biology is the need to distinguish indolent from aggressive disease. Elucidating the molecular determinants of aggressiveness has major implications for developing new genomic diagnostics for clinicians to use, and epigenetic regulation of gene expression that results in more aggressive cancers are compelling drug targets. Here, we hypothesize that differential DNA methylation in gene bodies regulates the production of tumor-promoting alternative transcription events in aggressive prostate cancer. DNA methylation may serve a regulatory role in concert with regulatory proteins and sequence features because transcription start site selection and alternative splicing occur co-transcriptionally. To detect interdependencies between differential DNA methylation and alternative isoforms, we utilized RNA-seq and Illumina HumanMethylation 450K array data produced by The Cancer Genome Atlas for 537 normal prostate and prostate cancer tissue specimens. Reference-guided de novo transcriptome assembly was performed, and fractional usage was computed at both the exon and isoform level for 4,734 expressed genes that contain gene-body differential methylation sites on the 450K array. We identified a set of 120 genes with correlations (|Spearman correlation| > 0.5, FDR adj. p-value < 0.05 and difference > 5% between fractional usage when grouped by the top and bottom quintile of DNA methylation) between the exon or isoform usage fraction and gene body CpG site methylation level. Of these, 53 genes contain differential DNA methylation and exon or isoform usage specific to aggressive disease. This gene set includes SEPT9, which is known to have a cell migration promoting alternative promoter regulated by with DNA methylation changes in breast cancer, as well as two genes with prior links to prostate cancer aggressiveness (MT2A and TDRD1). At the exon level, there were 6 cases where at least one differential methylation site associated with first exon usage, 7 with last exons, and 121 with internal exons. At the isoform level, 92 isoforms that associated with gene body DNA methylation contained alternative promoter usage, 80 contained alternative end usage, 71 contained alternative splice donor/acceptor sites, and 82 contained exon skipping/inclusion events. Additionally, 4 isoforms were predicted to be targets for nonsense mediated decay (NMD) by inducing message frameshifts that reveal internal stop codons. The diversity of this distribution suggests that DNA methylation may interact with a variety of different regulatory mechanisms that occur co-transcriptionally. While the 450K array only covers a small fraction of gene body CpG sites, our analysis supports the possibility of alternative isoform usage regulated by prostate cancer-specific and aggressiveness subtype-specific DNA methylation changes. Future work will interrogate the exact nature of the mechanisms by which DNA methylation changes cross talk with regulatory factors in alternative splicing, promoter usage, and polyadenylation. Such mechanisms may be targetable pharmacologically for tumor suppressive effect. Citation Format: Jeffrey M. Bhasin, Angela H. Ting. Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A45.

Bioinformatic analysis often produces large sets of genomic ranges that can be difficult to interpret in the absence of genomic context. Goldmine annotates genomic ranges from any source with gene model and feature contexts to facilitate global descriptions and candidate loci discovery. We demonstrate the value of genomic context by using Goldmine to elucidate context dynamics in transcription factor binding and to reveal differentially methylated regions (DMRs) with context-specific functional correlations. The open source R package and documentation for Goldmine are available at http://jeffbhasin.github.io/goldmine.

Background: Fibrosis of the intestine is a common and poorly understood complication of Crohn's disease (CD) characterized by excessive deposition of extracellular matrix and accompanied by narrowing and obstruction of the gut lumen. Defining the molecular characteristics of this fibrotic disorder is a vital step in the development of specific prediction, prevention, and treatment strategies. Previous epigenetic studies indicate that alterations in DNA methylation could explain the mechanism by which mesenchymal cells adopt the requisite pro-fibrotic phenotype that promotes fibrosis progression. However, to date, genome-wide analysis of the DNA methylome of any type of human fibrosis is lacking. We employed an unbiased approach using deep sequencing to define the DNA methylome and transcriptome of purified fibrotic human intestinal fibroblasts (HIF) from the colons of patients with fibrostenotic CD.Results: When compared with normal fibroblasts, we found that the majority of differential DNA methylation was within introns and intergenic regions and not associated with CpG islands. Only a low percentage occurred in the promoters and exons of genes. Integration of the DNA methylome and transcriptome identified regions in three genes that inversely correlated with gene expression: wingless-type mouse mammary tumor virus integration site family, member 2B (WNT2B) and two eicosanoid synthesis pathway enzymes (prostacyclin synthase and prostaglandin D2 synthase). These findings were independently validated by RT-PCR and bisulfite sequencing. Network analysis of the data also identified candidate molecular interactions relevant to fibrosis pathology.Conclusions: Our definition of a genome-wide fibrosis-specific DNA methylome provides new gene networks and epigenetic states by which to understand mechanisms of pathological gene expression that lead to fibrosis. Our data also provide a basis for development of new fibrosis-specific therapies, as genes dysregulated in fibrotic Crohn's disease, following functional validation, can serve as new therapeutic targets.

DNA methylation differences capture substantial information about the molecular and gene-regulatory states among biological subtypes. Enrichment-based next generation sequencing methods such as MBD-isolated genome sequencing (MiGS) and MeDIP-seq are appealing for studying DNA methylation genome-wide in order to distinguish between biological subtypes. However, current analytic tools do not provide optimal features for analyzing three-group or larger study designs. MethylAction addresses this need by detecting all possible patterns of statistically significant hyper- and hypo- methylation in comparisons involving any number of groups. Crucially, significance is established at the level of differentially methylated regions (DMRs), and bootstrapping determines false discovery rates (FDRs) associated with each pattern. We demonstrate this functionality in a four-group comparison among benign prostate and three clinical subtypes of prostate cancer and show that the bootstrap FDRs are highly useful in selecting the most robust patterns of DMRs. Compared to existing tools that are limited to two-group comparisons, MethylAction detects more DMRs with strong differential methylation measurements confirmed by whole genome bisulfite sequencing and offers a better balance between precision and recall in cross-cohort comparisons. MethylAction is available as an R package at http://jeffbhasin.github.io/methylaction.

A critical need in understanding the biology of prostate cancer is characterizing the molecular differences between indolent and aggressive cases. Because DNA methylation can capture the regulatory state of tumors, we analyzed differential methylation patterns genome-wide among benign prostatic tissue and low-grade and high-grade prostate cancer and found extensive, focal hypermethylation regions unique to high-grade disease. These hypermethylation regions occurred not only in the promoters of genes but also in gene bodies and at intergenic regions that are enriched for DNA-protein binding sites. Integration with existing RNA-sequencing (RNA-seq) and survival data revealed regions where DNA methylation correlates with reduced gene expression associated with poor outcome. Regions specific to aggressive disease are proximal to genes with distinct functions from regions shared by indolent and aggressive disease. Our compendium of methylation changes reveals crucial molecular distinctions between indolent and aggressive prostate cancer.

A major challenge in the clinical management of prostate cancer is the inability to definitively diagnose indolent versus aggressive cases. Contributing to this challenge is a lack of basic science understanding of the molecular basis behind aggressiveness subtypes in prostate cancer. DNA methylation is the epigenetic addition of a methyl group to the DNA base cytosine and has been found to regulate cell proliferation and environmental adaptation. We hypothesized that DNA methylation changes are a mechanism by which an aggressive cancer attains phenotypes that distinguish it from indolent cases via disruption of regulatory networks. This hypothesis was tested by comparing DNA methylation between benign prostate and both low grade (Gleason score 6) and high grade (Gleason score 8 to 10) groups. Methylome-wide next generation sequencing was performed on formalin-fixed paraffin embedded (FFPE) samples from radical prostatectomy cases using MBD-isolated genome sequencing (MiGS). Global clustering analyses showed some separation between the three groups, but also indicated some heterogeneity. Specific differentially methylated regions (DMRs) were detected between the three groups. The two most prevalent were hypermethylation specific to high grade (491 DMRs at 4% false discovery rate) and hypermethylation shared between both cancer subtypes (1334 DMRs at 1% false discovery rate). Statistical and computational analysis of this data set was non-trivial and the software developed to perform the analysis has been made available. High grade specific DMRs are abundant in intergenic and gene body contexts, and occur less frequently in promoters and CpG islands than DMRs shared between both subtypes. Intergenic DMRs are enriched for putative functional elements (ChIP-seq peaks and DNaseI hypersensitive sites from the ENCODE project) suggesting that these changes may have functional impacts as local or regional enhancers regulating multiple genes. To analyze this in more detail, DNA-protein binding sites for specific factors were compared between 5 spatial contexts. Differential patterns of enrichment were observed for regulatory binding factors between promoters, intergenic regions, and gene 39 ends. A de novo motif search revealed 8 frequent sequence motifs in intergenic DMRs that are not similar to any known binding motifs, indicating possible binding sites for as yet unknown factors. For DMRs that are proximal to genes, an interaction network based on curated pathway data was built using the GeneMANIA Cytoscape plugin. Gene ontology enrichment was observed for “positive regulation of cell motility” (Q-value=1.4e-07, 22 of 296 genes in term) and “regulatory region DNA binding” (Q-value=6.7e-07, 18 of 255 genes in term) within this network. Interestingly, each GO term appears as a distinct cluster on the interaction network. While the specific regulatory action of these DMRs and exact target genes in aggressive cancer remains to be determined experimentally, this work has established the presence of focal hypermethylation that distinguishes indolent and aggressive prostate cancer. The high frequency of intergenic but functionally enriched genomic contexts and functional enrichment of these DMRs at genic sites for transcription factors suggests that they regulate aggressiveness in a complex manner through secondary and tertiary effects. This study provides a first glimpse at molecular differences between indolent and aggressive prostate and future work may lead to the development of biomarkers and treatments based on this knowledge. Citation Format: Jeffrey M. Bhasin, Lars Matkin, Margaret G. Taylor, Byron H. Lee, Cristina Magi-Galluzi, Eric A. Klein, Bo Hu, Yaomin Xu, Angela H. Ting. DNA methylation sequencing of prostate tumors: Possible molecular mechanisms for cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-24.

Background A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs) for complex traits are sex specific, but the reason for this is not well understood. Methodology/Principal Findings RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F2 mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs) were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only ∼1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing. Conclusions/Significance These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific.

Objective - Apolipoprotein ( apo) E-deficient mice are hypercholesterolemic and develop atherosclerosis on low-fat chow diets; however, the genetic background strain has a large effect on atherosclerosis susceptibility. This study aimed to determine the genetic regions associated with strain effects on lesion area.Methods and Results - We performed a strain intercross between atherosclerosis sensitive DBA/2 and atherosclerosis resistant AKR apoE-deficient mice. Aortic root lesion area, total cholesterol, body weights, and complete blood counts were ascertained for 114 male and 95 female F-2 progeny. A high-density genome scan was performed using a mouse single nucleotide polymorphism chip yielding 1967 informative polymorphic markers. Quantitative trait locus ( QTL) statistical analyses were performed. Novel loci associated with lesion or log lesion area were identified for the female and male F2 cohorts. The atherosclerosis QTLs in female mice reside on chromosomes 15, 5, 3, and 13, and in male mice on chromosomes 17, 18, and 2. QTL were also identified for body weight, total cholesterol, and blood count parameters.Conclusions - Loci were identified for atherosclerosis susceptibility in a strain intercross study. The identity of the responsible genes at these loci remains to be determined.