Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling.

BACKGROUND: The Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples. RESULTS: We find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer's recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2's new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding. CONCLUSIONS: Overall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

MethPanel: a parallel pipeline and interactive analysis tool for multiplex bisulphite PCR sequencing to assess DNA methylation biomarker panels for disease detection.

SUMMARY: DNA methylation patterns in a cell are associated with gene expression and the phenotype of a cell, including disease states. Bisulphite PCR sequencing is commonly used to assess the methylation profile of genomic regions between different cells. Here we have developed MethPanel, a computational pipeline with an interactive graphical interface to rapidly analyse multiplex bisulphite PCR sequencing data. MethPanel comprises a complete analysis workflow from genomic alignment to DNA methylation calling and supports an unlimited number of PCR amplicons and input samples. MethPanel offers important and unique features, such as calculation of an epipolymorphism score and bisulphite PCR bias correction capabilities, and is designed so that the methylation data from all samples can be processed in parallel. The outputs are automatically forwarded to a shinyApp for convenient display, visualization and remotely sharing data with collaborators and clinicians. AVAILABILITYAND IMPLEMENTATION: MethPanel is freely available at https://github.com/thinhong/MethPanel. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enduring epigenetic landmarks define the cancer microenvironment.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Evaluation of cross-platform and interlaboratory concordance via consensus modelling of genomic measurements.

MOTIVATION: A synoptic view of the human genome benefits chiefly from the application of nucleic acid sequencing and microarray technologies. These platforms allow interrogation of patterns such as gene expression and DNA methylation at the vast majority of canonical loci, allowing granular insights and opportunities for validation of original findings. However, problems arise when validating against a "gold standard" measurement, since this immediately biases all subsequent measurements towards that particular technology or protocol. Since all genomic measurements are estimates, in the absence of a "gold standard" we instead empirically assess the measurement precision and sensitivity of a large suite of genomic technologies via a consensus modelling method called the row-linear model. This method is an application of the American Society for Testing and Materials Standard E691 for assessing interlaboratory precision and sources of variability across multiple testing sites. Both cross-platform and cross-locus comparisons can be made across all common loci, allowing identification of technology- and locus-specific tendencies. RESULTS: We assess technologies including the Infinium MethylationEPIC BeadChip, whole genome bisulfite sequencing (WGBS), two different RNA-Seq protocols (PolyA+ and Ribo-Zero) and five different gene expression array platforms. Each technology thus is characterised herein, relative to the consensus. We showcase a number of applications of the row-linear model, including correlation with known interfering traits. We demonstrate a clear effect of cross-hybridisation on the sensitivity of Infinium methylation arrays. Additionally, we perform a true interlaboratory test on a set of samples interrogated on the same platform across twenty-one separate testing laboratories. AVAILABILITY AND IMPLEMENTATION: A full implementation of the row-linear model, plus extra functions for visualisation, are found in the R package consensus at https://github.com/timpeters82/consensus. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Schizophrenia-associated methylomic variation: molecular signatures of disease and polygenic risk burden across multiple brain regions.

Genetic association studies provide evidence for a substantial polygenic component to schizophrenia, although the neurobiological mechanisms underlying the disorder remain largely undefined. Building on recent studies supporting a role for developmentally regulated epigenetic variation in the molecular aetiology of schizophrenia, this study aimed to identify epigenetic variation associated with both a diagnosis of schizophrenia and elevated polygenic risk burden for the disease across multiple brain regions. Genome-wide DNA methylation was quantified in 262 post-mortem brain samples, representing tissue from four brain regions (prefrontal cortex, striatum, hippocampus and cerebellum) from 41 schizophrenia patients and 47 controls. We identified multiple disease-associated and polygenic risk score-associated differentially methylated positions and regions, which are not enriched in genomic regions identified in genetic studies of schizophrenia and do not reflect direct genetic effects on DNA methylation. Our study represents the first analysis of epigenetic variation associated with schizophrenia across multiple brain regions and highlights the utility of polygenic risk scores for identifying molecular pathways associated with aetiological variation in complex disease.

DNA methylation profiles in preeclampsia and healthy control placentas.

Preeclampsia is a hypertensive disorder of pregnancy that affects 3-5% of all pregnancies. There is evidence to suggest that epigenetic mechanisms, such as DNA methylation, play a role in placental development and function. This study compared DNA methylation profiles of placentas from preeclampsia-affected pregnancies with placentas from healthy pregnancies to identify gene-specific changes in DNA methylation that may contribute to the development of preeclampsia. The methylation status of eight placental biopsies taken from preeclampsia-affected and 16 healthy pregnancies was analyzed using the Illumina Infinium Methylation 450 BeadChip array. Bisulfite pyrosequencing was used to confirm regions found to be differentially methylated between preeclampsia and healthy placentas. A total of 303 differentially methylated regions, 214 hypermethylated and 89 hypomethylated, between preeclampsia cases and controls were identified, after adjusting for gestational age (adjusted P < 0.05). Functional annotation found cell adhesion, wingless type MMTV Integration Site family member 2 (Wnt) signaling pathway, and regulation of transcription were significantly enriched in these gene regions. Hypermethylation of WNT2, sperm equatorial segment protein (SPESP1), NADPH oxidase 5 (NOX5), and activated leukocyte cell adhesion molecule (ALCAM) in preeclampsia placentas was confirmed with pyrosequencing. This study found differences in methylation in gene regions involved in cell signaling (WNT2), fertilization and implantation (SPESP1), reactive oxygen species signaling (NOX5), and cell adhesion (ALCAM). These results build on recently published studies that have reported significant differences in DNA methylation in preeclampsia placentas.

Mutation of the diamond-blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes.

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

Epigenome-wide scans identify differentially methylated regions for age and age-related phenotypes in a healthy ageing population.

Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype-phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

A data-driven approach to preprocessing Illumina 450K methylation array data.

BACKGROUND: As the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the research community. The landscape of DNA methylation across tissues, through development and in disease pathogenesis is not yet well characterized. Thus there is a need for rapid and cost effective methods for assessing genome-wide levels of DNA methylation. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a very useful addition to the available methods for DNA methylation analysis but its complex design, incorporating two different assay methods, requires careful consideration. Accordingly, several normalization schemes have been published. We have taken advantage of known DNA methylation patterns associated with genomic imprinting and X-chromosome inactivation (XCI), in addition to the performance of SNP genotyping assays present on the array, to derive three independent metrics which we use to test alternative schemes of correction and normalization. These metrics also have potential utility as quality scores for datasets. RESULTS: The standard index of DNA methylation at any specific CpG site is ß = M/(M + U + 100) where M and U are methylated and unmethylated signal intensities, respectively. Betas (ßs) calculated from raw signal intensities (the default GenomeStudio behavior) perform well, but using 11 methylomic datasets we demonstrate that quantile normalization methods produce marked improvement, even in highly consistent data, by all three metrics. The commonly used procedure of normalizing betas is inferior to the separate normalization of M and U, and it is also advantageous to normalize Type I and Type II assays separately. More elaborate manipulation of quantiles proves to be counterproductive. CONCLUSIONS: Careful selection of preprocessing steps can minimize variance and thus improve statistical power, especially for the detection of the small absolute DNA methylation changes likely associated with complex disease phenotypes. For the convenience of the research community we have created a user-friendly R software package called wateRmelon, downloadable from bioConductor, compatible with the existing methylumi, minfi and IMA packages, that allows others to utilize the same normalization methods and data quality tests on 450K data.

Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder.

Studies of the major psychoses, schizophrenia (SZ) and bipolar disorder (BD), have traditionally focused on genetic and environmental risk factors, although more recent work has highlighted an additional role for epigenetic processes in mediating susceptibility. Since monozygotic (MZ) twins share a common DNA sequence, their study represents an ideal design for investigating the contribution of epigenetic factors to disease etiology. We performed a genome-wide analysis of DNA methylation on peripheral blood DNA samples obtained from a unique sample of MZ twin pairs discordant for major psychosis. Numerous loci demonstrated disease-associated DNA methylation differences between twins discordant for SZ and BD individually, and together as a combined major psychosis group. Pathway analysis of our top loci highlighted a significant enrichment of epigenetic changes in biological networks and pathways directly relevant to psychiatric disorder and neurodevelopment. The top psychosis-associated, differentially methylated region, significantly hypomethylated in affected twins, was located in the promoter of ST6GALNAC1 overlapping a previously reported rare genomic duplication observed in SZ. The mean DNA methylation difference at this locus was 6%, but there was considerable heterogeneity between families, with some twin pairs showing a 20% difference in methylation. We subsequently assessed this region in an independent sample of postmortem brain tissue from affected individuals and controls, finding marked hypomethylation (>25%) in a subset of psychosis patients. Overall, our data provide further evidence to support a role for DNA methylation differences in mediating phenotypic differences between MZ twins and in the etiology of both SZ and BD.

A first-in-class pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition.

Cyclin-dependent kinase 5 is activated by small subunits, of which p35 is the most abundant. The functions of cyclin-dependent kinase 5 signalling in cognition and cognitive disorders remains unclear. Here, we show that in schizophrenia, a disorder associated with impaired cognition, p35 expression is reduced in relevant brain regions. Additionally, the expression of septin 7 and OPA1, proteins downstream of truncated p35, is decreased in schizophrenia. Mimicking a reduction of p35 in heterozygous knockout mice is associated with cognitive endophenotypes. Furthermore, a reduction of p35 in mice results in protein changes similar to schizophrenia post-mortem brain. Hence, heterozygous p35 knockout mice model both cognitive endophenotypes and molecular changes reminiscent of schizophrenia. These changes correlate with reduced acetylation of the histone deacetylase 1 target site H3K18 in mice. This site has previously been shown to be affected by truncated p35. By restoring H3K18 acetylation with the clinically used specific histone deacetylase 1 inhibitor MS-275 both cognitive and molecular endophenotypes of schizophrenia can be rescued in p35 heterozygous knockout mice. In summary, we suggest that reduced p35 expression in schizophrenia has an impact on synaptic protein expression and cognition and that these deficits can be rescued, at least in part, by the inhibition of histone deacetylase 1.

Comprehensive methylome sequencing reveals prognostic epigenetic biomarkers for prostate cancer mortality.

BACKGROUND: Prostate cancer is a clinically heterogeneous disease with a subset of patients rapidly progressing to lethal-metastatic prostate cancer. Current clinicopathological measures are imperfect predictors of disease progression. Epigenetic changes are amongst the earliest molecular changes in tumourigenesis. To find new prognostic biomarkers to enable earlier intervention and improved outcomes, we performed methylome sequencing of DNA from patients with localised prostate cancer and long-term clinical follow-up. METHODS: We used whole-genome bisulphite sequencing (WGBS) to comprehensively map and compare DNA methylation of radical prostatectomy tissue between patients with lethal disease (n = 7) and non-lethal (n = 8) disease (median follow-up 19.5 years). Validation of differentially methylated regions (DMRs) was performed in an independent cohort (n = 185, median follow-up 15 years) using targeted multiplex bisulphite sequencing of candidate regions. Survival was assessed via univariable and multivariable analyses including clinicopathological measures (log-rank and Cox regression models). RESULTS: WGBS data analysis identified cancer-specific methylation patterns including CpG island hypermethylation, and hypomethylation of repetitive elements, with increasing disease risk. We identified 1420 DMRs associated with prostate cancer-specific mortality (PCSM), which showed enrichment for gene sets downregulated in prostate cancer and de novo methylated in cancer. Through comparison with public prostate cancer datasets, we refined the DMRs to develop an 18-gene prognostic panel. Applying this panel to an independent cohort, we found significant associations between PCSM and hypermethylation at EPHB3, PARP6, TBX1, MARCH6 and a regulatory element within CACNA2D4. Strikingly in a multivariable model, inclusion of CACNA2D4 methylation was a better predictor of PCSM versus grade alone (Harrell's C-index: 0.779 vs. 0.684). CONCLUSIONS: Our study provides detailed methylome maps of non-lethal and lethal prostate cancer and identifies novel genic regions that distinguish these patient groups. Inclusion of our DNA methylation biomarkers with existing clinicopathological measures improves prognostic models of prostate cancer mortality, and holds promise for clinical application.

Identification of DNA methylation biomarkers with potential to predict response to neoadjuvant chemotherapy in triple-negative breast cancer.

Neoadjuvant chemotherapy (NAC) is used to treat triple-negative breast cancer (TNBC) prior to resection. Biomarkers that accurately predict a patient's response to NAC are needed to individualise therapy and avoid chemotoxicity from unnecessary chemotherapy. We performed whole-genome DNA methylation profiling on diagnostic TNBC biopsy samples from the Sequential Evaluation of Tumours Undergoing Preoperative (SETUP) NAC study. We found 9 significantly differentially methylated regions (DMRs) at diagnosis which were associated with response to NAC. We show that 4 of these DMRs are associated with TNBC overall survival (P < 0.05). Our results highlight the potential of DNA methylation biomarkers for predicting NAC response in TNBC.

The role of Tuba1a in adult hippocampal neurogenesis and the formation of the dentate gyrus.

The multitubulin hypothesis holds that each tubulin isotype serves a unique role with respect to microtubule function. Here we investigate the role of the α-tubulin subunit Tuba1a in adult hippocampal neurogenesis and the formation of the dentate gyrus. Employing birth date labelling and immunohistological markers, we show that mice harbouring an S140G mutation in Tuba1a present with normal neurogenic potential, but that this neurogenesis is often ectopic. Morphological analysis of the dentate gyrus in adulthood revealed a disorganised subgranular zone and a dispersed granule cell layer. We have shown that these anatomical abnormalities are due to defective migration of prospero-homeobox-1-positive neurons and T-box-brain-2-positive progenitors during development. Such migratory defects may also be responsible for the cytoarchitectural defects observed in the dentate gyrus of patients with mutations in TUBA1A.

Advances in Prognostic Methylation Biomarkers for Prostate Cancer.

There is a major clinical need for accurate biomarkers for prostate cancer prognosis, to better inform treatment strategies and disease monitoring. Current clinically recognised prognostic factors, including prostate-specific antigen (PSA) levels, lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised intermediate grade prostate cancer. There has therefore been a major focus on identifying molecular biomarkers that can add prognostic value to existing markers, including investigation of DNA methylation, which has a known role in tumorigenesis. In this review, we will provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis, and highlight the advances that have been made in this field. We cover the numerous studies into well-established candidate genes, and explore the technological transition that has enabled hypothesis-free genome-wide studies and the subsequent discovery of novel prognostic genes.

Alterations in the methylome of the stromal tumour microenvironment signal the presence and severity of prostate cancer.

BACKGROUND: Prostate cancer changes the phenotype of cells within the stromal microenvironment, including fibroblasts, which in turn promote tumour progression. Functional changes in prostate cancer-associated fibroblasts (CAFs) coincide with alterations in DNA methylation levels at loci-specific regulatory regions. Yet, it is not clear how these methylation changes compare across CAFs from different patients. Therefore, we examined the consistency and prognostic significance of genome-wide DNA methylation profiles between CAFs from patients with different grades of primary prostate cancer. RESULTS: We used Infinium MethylationEPIC BeadChips to evaluate genome-wide DNA methylation profiles from 18 matched CAFs and non-malignant prostate tissue fibroblasts (NPFs) from men with moderate to high grade prostate cancer, as well as five unmatched benign prostate tissue fibroblasts (BPFs) from men with benign prostatic hyperplasia. We identified two sets of differentially methylated regions (DMRs) in patient CAFs. One set of DMRs reproducibly differed between CAFs and fibroblasts from non-malignant tissue (NPFs and BPFs). Indeed, more than 1200 DMRs consistently changed in CAFs from every patient, regardless of tumour grade. The second set of DMRs varied between CAFs according to the severity of the tumour. Notably, hypomethylation of the EDARADD promoter occurred specifically in CAFs from high-grade tumours and correlated with increased transcript abundance and increased EDARADD staining in patient tissue. Across multiple cohorts, tumours with low EDARADD DNA methylation and high EDARADD mRNA expression were consistently associated with adverse clinical features and shorter recurrence free survival. CONCLUSIONS: We identified a large set of DMRs that are commonly shared across CAFs regardless of tumour grade and outcome, demonstrating highly consistent epigenome changes in the prostate tumour microenvironment. Additionally, we found that CAFs from aggressive prostate cancers have discrete methylation differences compared to CAFs from moderate risk prostate cancer. Together, our data demonstrates that the methylome of the tumour microenvironment reflects both the presence and the severity of the prostate cancer and, therefore, may provide diagnostic and prognostic potential.