Application of deep learning in cancer epigenetics through DNA methylation analysis.

DNA methylation is a fundamental epigenetic modification involved in various biological processes and diseases. Analysis of DNA methylation data at a genome-wide and high-throughput level can provide insights into diseases influenced by epigenetics, such as cancer. Recent technological advances have led to the development of high-throughput approaches, such as genome-scale profiling, that allow for computational analysis of epigenetics. Deep learning (DL) methods are essential in facilitating computational studies in epigenetics for DNA methylation analysis. In this systematic review, we assessed the various applications of DL applied to DNA methylation data or multi-omics data to discover cancer biomarkers, perform classification, imputation and survival analysis. The review first introduces state-of-the-art DL architectures and highlights their usefulness in addressing challenges related to cancer epigenetics. Finally, the review discusses potential limitations and future research directions in this field.

Exploring Potential Epigenetic Biomarkers for Colorectal Cancer Metastasis.

Metastatic progression is a complex, multistep process and the leading cause of cancer mortality. There is growing evidence that emphasises the significance of epigenetic modification, specifically DNA methylation and histone modifications, in influencing colorectal (CRC) metastasis. Epigenetic modifications influence the expression of genes involved in various cellular processes, including the pathways associated with metastasis. These modifications could contribute to metastatic progression by enhancing oncogenes and silencing tumour suppressor genes. Moreover, specific epigenetic alterations enable cancer cells to acquire invasive and metastatic characteristics by altering cell adhesion, migration, and invasion-related pathways. Exploring the involvement of DNA methylation and histone modification is crucial for identifying biomarkers that impact cancer prediction for metastasis in CRC. This review provides a summary of the potential epigenetic biomarkers associated with metastasis in CRC, particularly DNA methylation and histone modifications, and examines the pathways associated with these biomarkers.

The transposable element-derived transcript of LIN28B has a placental origin and is not specific to tumours.

Transposable elements (TEs) are genetic elements that have evolved as crucial regulators of human development and cancer, functioning as both genes and regulatory elements. When TEs become dysregulated in cancer cells, they can serve as alternate promoters to activate oncogenes, a process known as onco-exaptation. This study aimed to explore the expression and epigenetic regulation of onco-exaptation events in early human developmental tissues. We discovered co-expression of some TEs and oncogenes in human embryonic stem cells and first trimester and term placental tissues. Previous studies identified onco-exaptation events in various cancer types, including an AluJb SINE element-LIN28B interaction in lung cancer cells, and showed that the TE-derived LIN28B transcript is associated with poor patient prognosis in hepatocellular carcinoma. This study further characterized the AluJb-LIN28B transcript and confirmed that its expression is restricted to the placenta. Targeted DNA methylation analysis revealed differential methylation of the two LIN28B promoters between placenta and healthy somatic tissues, indicating that some TE-oncogene interactions are not cancer-specific but arise from the epigenetic reactivation of developmental TE-derived regulatory events. In conclusion, our findings provide evidence that some TE-oncogene interactions are not limited to cancer and may originate from the epigenetic reactivation of TE-derived regulatory events that are involved in early development. These insights broaden our understanding of the role of TEs in gene regulation and suggest the potential importance of targeting TEs in cancer therapy beyond their conventional use as cancer-specific markers.

DNA methylome in pancreatic cancer identified novel promoter hyper-methylation in NPY and FAIM2 genes associated with poor prognosis in Indian patient cohort.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading cancers worldwide and has a poor survival, with a 5-year survival rate of only 8.5%. In this study we investigated altered DNA methylation associated with PDAC severity and prognosis. METHODS: Methylome data, generated using 450 K bead array, was compared between paired PDAC and normal samples in the TCGA cohort (n = 9) and our Indian cohort (n = 7). The total Indian Cohort (n = 75) was split into cohort 1 (n = 7), cohort 2 (n = 22), cohort 3 (n = 26) and cohort 4 (n = 20).Validation of differential methylation (6 selected CpG loci) and associated gene expression for differentially methylated genes (10 selected gDMs) were carried out in separate validation cohorts, using MSP, RT-PCR and IHC correlations between methylation and gene expression were observed in TCGA, GTEx cohorts and in validation cohorts. Kaplan-Meier survival analysis was done to study differential prognosis, during 2-5 years of follow-up. RESULTS: We identified 156 DMPs, mapped to 91 genes (gDMs), in PDAC; 68 (43.5%) DMPs were found to be differentially methylated both in TCGA cohort and our cohort, with significant concordance at hypo- and hyper-methylated loci. Enrichments of "regulation of ion transport", "Interferon alpha/beta signalling", "morphogenesis and development" and "transcriptional dysregulation" pathways were observed among 91 gDMs. Hyper-methylation of NPY and FAIM2 genes with down-regulated expression in PDAC, were significantly associated with poor prognosis in the Indian patient cohort. CONCLUSIONS: Ethnic variations among populations may determine the altered epigenetic landscape in the PDAC patients of the Indian cohort. Our study identified novel differentially methylated genes (mainly NPY and FAIM2) and also validated the previously identified differentially methylated CpG sites associated with PDAC cancer patient's survival. Comparative analysis of our data with TCGA and CPTAC cohorts showed that both NPY and FAIM2 hyper-methylation and down-regulations can be novel epigenetically regulated genes in the Indian patient population, statistically significantly associated with poor survival and advanced tumour stages.

Targeting DNA Methylation and EZH2 Activity to Overcome Melanoma Resistance to Immunotherapy.

Methylation of DNA at CpG sites is the most common and stable of epigenetic changes in cancer. Hypermethylation acts to limit immune checkpoint blockade immunotherapy by inhibiting endogenous interferon responses needed for recognition of cancer cells. By contrast, global hypomethylation results in the expression of programmed death ligand 1 (PD-L1) and inhibitory cytokines, accompanied by epithelial-mesenchymal changes that can contribute to immunosuppression. The drivers of these contrasting methylation states are not well understood. DNA methylation also plays a key role in cytotoxic T cell 'exhaustion' associated with tumor progression. We present an updated exploratory analysis of how DNA methylation may define patient subgroups and can be targeted to develop tailored treatment combinations to help improve patient outcomes.

Dynamic Epigenetic Changes during a Relapse and Recovery Cycle in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disease with variable severity. Patients experience frequent relapses where symptoms increase in severity, leaving them with a marked reduction in quality of life. Previous work has investigated molecular differences between ME/CFS patients and healthy controls, but not the dynamic changes specific to each individual patient. We applied precision medicine here to map genomic changes in two selected ME/CFS patients through a period that contained a relapse recovery cycle. DNA was isolated from two patients and a healthy age/gender matched control at regular intervals and captured the patient relapse in each case. Reduced representation DNA methylation sequencing profiles were obtained spanning the relapse recovery cycle. Both patients showed a significantly larger methylome variability (10-20-fold) through the period of sampling compared with the control. During the relapse, changes in the methylome profiles of the two patients were detected in regulatory-active regions of the genome that were associated, respectively, with 157 and 127 downstream genes, indicating disturbed metabolic, immune and inflammatory functions. Severe health relapses in the ME/CFS patients resulted in functionally important changes in their DNA methylomes that, while differing between the two patients, led to very similar compromised physiology. DNA methylation as a signature of disease variability in ongoing ME/CFS may have practical applications for strategies to decrease relapse frequency.

Recent Discoveries in Epigenetic Modifications of Polycystic Kidney Disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a heritable renal disease that results in end-stage kidney disease, due to the uncontrolled bilateral growth of cysts throughout the kidneys. While it is known that a mutation within a PKD-causing gene is required for the development of ADPKD, the underlying mechanism(s) causing cystogenesis and progression of the disease are not well understood. Limited therapeutic options are currently available to slow the rate of cystic growth. Epigenetic modifications, including DNA methylation, are known to be altered in neoplasia, and several FDA-approved therapeutics target these disease-specific changes. As there are many similarities between ADPKD and neoplasia, we (and others) have postulated that ADPKD kidneys contain alterations to their epigenetic landscape that could be exploited for future therapeutic discovery. Here we summarise the current understanding of epigenetic changes that are associated with ADPKD, with a particular focus on the burgeoning field of ADPKD-specific alterations in DNA methylation.

Epigenetic drivers of tumourigenesis and cancer metastasis.

Since the completion of the first human genome sequence and the advent of next generation sequencing technologies, remarkable progress has been made in understanding the genetic basis of cancer. These studies have mainly defined genetic changes as either causal, providing a selective advantage to the cancer cell (a driver mutation) or consequential with no selective advantage (not directly causal, a passenger mutation). A vast unresolved question is how a primary cancer cell becomes metastatic and what are the molecular events that underpin this process. However, extensive sequencing efforts indicate that mutation may not be a causal factor for primary to metastatic transition. On the other hand, epigenetic changes are dynamic in nature and therefore potentially play an important role in determining metastatic phenotypes and this area of research is just starting to be appreciated. Unlike genetic studies, current limitations in studying epigenetic events in cancer metastasis include a lack of conceptual understanding and an analytical framework for identifying putative driver and passenger epigenetic changes. In this review, we discuss the key concepts involved in understanding the role of epigenetic alterations in the metastatic cascade. We particularly focus on driver epigenetic events, and we describe analytical approaches and biological frameworks for distinguishing between "epi-driver" and "epi-passenger" events in metastasis. Finally, we suggest potential directions for future research in this important area of cancer research.

The Genes of Life and Death: A Potential Role for Placental-Specific Genes in Cancer: Active retrotransposons in the placenta encode unique functional genes that may also be used by cancer cells to promote malignancy.

The placenta invades the adjacent uterus and controls the maternal immune system, like a cancer invades surrounding organs and suppresses the local immune response. Intriguingly, placental and cancer cells are globally hypomethylated and share an epigenetic phenomenon that is not well understood - they fail to silence repetitive DNA sequences (retrotransposons) that are silenced (methylated) in healthy somatic cells. In the placenta, hypomethylation of retrotransposons has facilitated the evolution of new genes essential for placental function. In cancer, hypomethylation is thought to contribute to activation of oncogenes, genomic instability, and retrotransposon unsilencing; the latter, we postulate, is possibly the most important consequence. Activation of placental retrotransposon-derived genes in cancer underpins our hypothesis that hypomethylation of these genes drives cancer cell invasion. This alludes to an interesting paradox, that while placental retrotransposon-derived genes are essential for promoting early hominid life, the same genes promote disease-susceptibility and death through cancer.

Genome-Scale Single Nucleotide Resolution Analysis of DNA Methylation in Human Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in the kidney and end stage renal disease by the fourth or fifth decade of life. Mutations in the PKD1 gene account for 85% of all cases of ADPKD. No curative therapy exists for patients affected by this disease and an underexplored avenue for the treatment of ADPKD is the targeting of epigenetic changes that occur during cystogenesis. Limited data exists on alterations in DNA methylation that are associated with ADPKD. Given the similarities between cyst growth and neoplasia, and the fact that 2 DNA methylation inhibitors are already Food and Drug Administration-approved for the treatment of myelodysplastic syndrome, we hypothesized that global DNA methylation patterns in ADPKD would parallel that observed in neoplasia, and which may provide an opportunity to treat ADPKD with epigenetic inhibitors. To address this hypothesis, we undertook a global DNA methylation analysis of human ADPKD kidney. METHODS: We generated single nucleotide resolution methylomes of cortical kidney tissue from individuals with ADPKD, and from non-ADPKD kidney tissue, using reduced representation bisulfite sequencing. Using quantitative reverse transcription polymerase chain reaction, we investigated expression of the PKD1 gene in both ADPKD and non-ADPKD kidney. RESULTS: We have shown that ADPKD-derived genomic DNA exhibits global hypomethylation when compared with non-ADPKD kidney, a pattern commonly observed in DNA methylation studies of tumor-derived tissue. We have also identified 13 discrete regions that are significantly differentially methylated in ADPKD compared to non-ADPKD, and 8 of these are gene specific. The PKD1 gene shows an increase in methylation at the 3' end of the gene body, but in contrast to previously published data, this is not associated with a decrease in PKD1 mRNA expression. CONCLUSION: This genome-scale single nucleotide resolution analysis of DNA methylation in human polycystic kidneys suggests that DNA methylation differences at specific loci are associated with ADPKD. Further exploration into the significance of these preliminary results may shed light on the disease process, and potentially reveal new therapeutic possibilities.

All-Trans Retinoic Acid Ameliorates Arsenic-Induced Oxidative Stress and Apoptosis in the Rat Uterus by Modulating MAPK Signaling Proteins.

Exposure to arsenic leads to inhibition of the anti-oxidant defense mechanism of the body. Reactive oxygen species generated in response to arsenic causes reproductive failures in exposed females and also acts as an inducer of apoptosis. As a prospective remedial agent, all-trans retinoic acid (ATRA) was assessed for reversing arsenic-induced oxidative stress and apoptosis. Rats exposed to arsenic for 28 days were allowed to recover naturally or were treated simultaneously with ATRA for 28 days or up to 56 days. Production of H2 O2 was detected using 2',7'-dichlorfluorescein diacetate (DCFCA) by flow cytometry. Catalase, superoxide dismutase, glutathione, ALT, and AST were estimated by biochemical assays and Western blot analyses. Detection of apoptosis was performed using annexin V-FITC/propidium iodide. Expressions of p53, p21, cleaved caspase 3, JNK/pJNK, and ERK/pERK levels were estimated using Western blot analysis. Elemental arsenic deposition in the rat uterus and liver was estimated by atomic absorption spectrophotometry. Our results confirmed that ATRA ameliorated sodium arsenite-induced ROS generation, restored redox balance, and prevented apoptosis. Concomitant recovery was observed to be more prominent for ATRA-treated rats as compared to the rats that were allowed to recover naturally for 56 days. Tissue arsenic deposition was significantly reduced in the uterus upon continuous ATRA treatment. The results revealed that ATRA reversed arsenic-induced free radical generation, activated the anti-oxidant defence system, and subsequently repressed p53-dependent apoptosis through inhibition of the MAPK signaling components. J. Cell. Biochem. 118: 3796-3809, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA expression patterns and signalling pathways in the development and progression of childhood solid tumours.

The development of childhood solid tumours is tied to early developmental processes. These tumours may be complex and heterogeneous, and elucidating the aberrant mechanisms that alter the early embryonic environment and lead to disease is essential to our understanding of how these tumours function. MicroRNAs (miRNAs) are vital regulators of gene expression at all stages of development, and their crosstalk via developmental signalling pathways is essential for orchestrating regulatory control in processes such as proliferation, differentiation and apoptosis of cells. Oncogenesis, from aberrant miRNA expression, can occur through amplification and overexpression of oncogenic miRNAs (oncomiRs), genetic loss of tumour suppressor miRNAs, and global miRNA reduction from genetic and epigenetic alterations in the components regulating miRNA biogenesis. While few driver mutations have been identified in many of these types of tumours, abnormal miRNA expression has been found in a number of childhood solid tumours compared to normal tissue. An exploration of the network of key developmental pathways and interacting miRNAs may provide insight into the development of childhood solid malignancies and how key regulators are affected. Here we present a comprehensive introduction to the roles and implications of miRNAs in normal early development and childhood solid tumours, highlighting several tumours in depth, including embryonal brain tumours, neuroblastoma, osteosarcoma, Wilms tumour, and hepatoblastoma. In light of recent literature describing newer classifications and subtyping of tumours based on miRNA profiling, we discuss commonly identified miRNAs, clusters or families associated with several solid tumours and future directions for improving therapeutic approaches.

DMAP: differential methylation analysis package for RRBS and WGBS data.

MOTIVATION: The rapid development of high-throughput sequencing technologies has enabled epigeneticists to quantify DNA methylation on a massive scale. Progressive increase in sequencing capacity present challenges in terms of processing analysis and the interpretation of the large amount of data; investigating differential methylation between genome-scale data from multiple samples highlights this challenge. RESULTS: We have developed a differential methylation analysis package (DMAP) to generate coverage-filtered reference methylomes and to identify differentially methylated regions across multiple samples from reduced representation bisulphite sequencing and whole genome bisulphite sequencing experiments. We introduce a novel fragment-based approach for investigating DNA methylation patterns for reduced representation bisulphite sequencing data. Further, DMAP provides the identity of gene and CpG features and distances to the differentially methylated regions in a format that is easily analyzed with limited bioinformatics knowledge. AVAILABILITY AND IMPLEMENTATION: The software has been implemented in C and has been written to ensure portability between different platforms. The source code and documentation is freely available (DMAP: as compressed TAR archive folder) from http://biochem.otago.ac.nz/research/databases-software/. Two test datasets are also available for download from the Web site. Test dataset 1 contains reads from chromosome 1 of a patient and a control, which is used for comparative analysis in the current article. Test dataset 2 contains reads from a part of chromosome 21 of three disease and three control samples for testing the operation of DMAP, especially for the analysis of variance. Example commands for the analyses are included.

Comparison of alignment software for genome-wide bisulphite sequence data.

Recent advances in next generation sequencing (NGS) technology now provide the opportunity to rapidly interrogate the methylation status of the genome. However, there are challenges in handling and interpretation of the methylation sequence data because of its large volume and the consequences of bisulphite modification. We sequenced reduced representation human genomes on the Illumina platform and efficiently mapped and visualized the data with different pipelines and software packages. We examined three pipelines for aligning bisulphite converted sequencing reads and compared their performance. We also comment on pre-processing and quality control of Illumina data. This comparison highlights differences in methods for NGS data processing and provides guidance to advance sequence-based methylation data analysis for molecular biologists.

Advances in single-cell long-read sequencing technologies.

With an increase in accuracy and throughput of long-read sequencing technologies, they are rapidly being assimilated into the single-cell sequencing pipelines. For transcriptome sequencing, these techniques provide RNA isoform-level information in addition to the gene expression profiles. Long-read sequencing technologies not only help in uncovering complex patterns of cell-type specific splicing, but also offer unprecedented insights into the origin of cellular complexity and thus potentially new avenues for drug development. Additionally, single-cell long-read DNA sequencing enables high-quality assemblies, structural variant detection, haplotype phasing, resolving high-complexity regions, and characterization of epigenetic modifications. Given that significant progress has primarily occurred in single-cell RNA isoform sequencing (scRiso-seq), this review will delve into these advancements in depth and highlight the practical considerations and operational challenges, particularly pertaining to downstream analysis. We also aim to offer a concise introduction to complementary technologies for single-cell sequencing of the genome, epigenome and epitranscriptome. We conclude by identifying certain key areas of innovation that may drive these technologies further and foster more widespread application in biomedical science.

Heat shock protein 90: biological functions, diseases, and therapeutic targets.

Heat shock protein 90 (Hsp90) is a predominant member among Heat shock proteins (HSPs), playing a central role in cellular protection and maintenance by aiding in the folding, stabilization, and modification of diverse protein substrates. It collaborates with various co-chaperones to manage ATPase-driven conformational changes in its dimer during client protein processing. Hsp90 is critical in cellular function, supporting the proper operation of numerous proteins, many of which are linked to diseases such as cancer, Alzheimer's, neurodegenerative conditions, and infectious diseases. Recognizing the significance of these client proteins across diverse diseases, there is a growing interest in targeting Hsp90 and its co-chaperones for potential therapeutic strategies. This review described biological background of HSPs and the structural characteristics of HSP90. Additionally, it discusses the regulatory role of heat shock factor-1 (HSF-1) in modulating HSP90 and sheds light on the dynamic chaperone cycle of HSP90. Furthermore, the review discusses the specific contributions of HSP90 in various disease contexts, especially in cancer. It also summarizes HSP90 inhibitors for cancer treatment, offering a thoughtful analysis of their strengths and limitations. These advancements in research expand our understanding of HSP90 and open up new avenues for considering HSP90 as a promising target for therapeutic intervention in a range of diseases.

Targeted DNA Methylation Editing Using an All-in-One System Establishes Paradoxical Activation of EBF3.

Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of the EBF3 promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.

Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

Genetic and epigenetic features of neuroendocrine prostate cancer and their emerging applications.

Prostate cancer is the second most prevalent cancer in men globally. De novo neuroendocrine prostate cancer (NEPC) is uncommon at initial diagnosis, however, (treatment-induced) t-NEPC emerges in up to 25% of prostate adenocarcinoma (PRAD) cases treated with androgen deprivation, carrying a drastically poor prognosis. The transition from PRAD to t-NEPC is underpinned by several key genetic mutations; TP53, RB1, and MYCN are the main genes implicated, bearing similarities to other neuroendocrine tumours. A broad range of epigenetic alterations, such as aberrations in DNA methylation, histone post-translational modifications, and non-coding RNAs, may drive lineage plasticity from PRAD to t-NEPC. The clinical diagnosis of NEPC is hampered by a lack of accessible biomarkers; recent advances in liquid biopsy techniques assessing circulating tumour cells and ctDNA in NEPC suggest that the advent of non-invasive means of monitoring progression to NEPC is on the horizon. Such techniques are vital for NEPC management; diagnosis of t-NEPC is crucial for implementing effective treatment, and precision medicine will be integral to providing the best outcomes for patients.

Cell-free DNA methylation in the clinical management of lung cancer.

The clinical use of cell-free DNA (cfDNA) methylation in managing lung cancer depends on its ability to differentiate between malignant and healthy cells, assign methylation changes to specific tissue sources, and elucidate opportunities for targeted therapy. From a technical standpoint, cfDNA methylation analysis is primed as a potential clinical tool for lung cancer screening, early diagnosis, prognostication, and treatment, pending the outcome of elaborate validation studies. Here, we discuss the current state of the art in cfDNA methylation analysis, examine the unique features and limitations of these new methods in a clinical context, propose two models for applying cfDNA methylation data for lung cancer screening, and discuss future research directions.