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Lavoro A, Ricci D, Gattuso G, Longo F, Spoto G, Vitale ACV, Giuliana MC, Falzone L, Libra M, Candido S. Recent advances on gene-related DNA methylation in cancer diagnosis, prognosis, and treatment: a clinical perspective. Clin Epigenetics 2025; 17:76. [PMID: 40325471 PMCID: PMC12054201 DOI: 10.1186/s13148-025-01884-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2025] [Accepted: 04/13/2025] [Indexed: 05/07/2025] Open
Abstract
Recent advances in screening programs and the development of innovative therapeutic strategies have significantly improved the clinical outcomes of cancer patients. However, many patients still experience treatment failure, primarily due to inherent or acquired drug resistance mechanisms. This challenge underscores the urgent need for novel therapeutic targets for the effective treatment of malignancies, as well as cancer-specific biomarkers to enhance early diagnosis and guide interventions. Epigenetic mechanisms, including DNA methylation, have recently garnered growing interest as key regulators of gene expression under both physiological and pathological conditions. Although epigenetic dysregulations are reliable tumor hallmarks, DNA methylation is still not routinely integrated into clinical practice, highlighting the need for further research to translate preclinical findings from the bench to the bedside. On these bases, the present review aims to illustrate the state of the art regarding the role of DNA methylation in cancer, describing the technologies currently available for DNA methylation profiling. Furthermore, the latest evidence on the application of DNA methylation hotspots in cancer diagnosis and prognosis, as well as the impact of epidrugs in cancer care, is discussed to provide a comprehensive overview of the potential clinical relevance of DNA methylation in advancing personalized medicine.
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Affiliation(s)
- Alessandro Lavoro
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | - Daria Ricci
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | - Giuseppe Gattuso
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | - Federica Longo
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | - Graziana Spoto
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | | | - Maria Chiara Giuliana
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
| | - Luca Falzone
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy.
| | - Massimo Libra
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
- Research Center for Prevention, Diagnosis and Treatment of Cancer, University of Catania, 95123, Catania, Italy
| | - Saverio Candido
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy
- Research Center for Prevention, Diagnosis and Treatment of Cancer, University of Catania, 95123, Catania, Italy
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2
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Nell RJ, Versluis M, Menger NV, Gelmi MC, Vu THK, Verdijk RM, Luyten GPM, Jager MJ, van der Velden PA. Digital PCR-based genetic profiling from vitreous fluid as liquid biopsy for primary uveal melanoma: a proof-of-concept study. J Exp Clin Cancer Res 2025; 44:124. [PMID: 40240901 PMCID: PMC12004579 DOI: 10.1186/s13046-025-03374-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2024] [Accepted: 03/25/2025] [Indexed: 04/18/2025] Open
Abstract
BACKGROUND Uveal melanoma is an aggressive ocular malignancy. Early molecular characterisation of primary tumours is crucial to identify those at risk of metastatic dissemination. Although tumour biopsies are being taken, liquid biopsies of ocular fluids may form a less invasive but relatively unexplored alternative. In this study, we aim to evaluate the DNA content of vitreous fluid from eyes with a uveal melanoma to obtain molecular tumour information. METHODS DNA was isolated from 65 vitreous fluid samples from enucleated eyes with a uveal melanoma and studied using digital PCR. Primary and additional driver mutations (in GNAQ, GNA11, PLCB4, CYSLTR2, BAP1, SF3B1 and EIF1AX) were investigated using accustomed targeted and drop-off assays. The copy numbers of chromosome 3p and 8q were measured using multiplex and single-nucleotide polymorphism-based assays. Our findings were compared to the molecular profile of matched primary tumours and to the clinicopathological tumour characteristics. RESULTS Almost all (63/65) vitreous fluids had measurable levels of DNA, but melanoma-cell derived DNA (containing the primary driver mutation) was detected in 45/65 samples (median proportion 15.5%, range 0.03-94.4%) and was associated with a larger tumour prominence, but not with any of the molecular tumour subtypes. Among the vitreous fluids with melanoma-cell derived DNA, not all samples harboured (analysable) other mutations or had sufficient statistical power to measure copy numbers. Still, additional mutations in BAP1, SF3B1 and EIF1AX were detected in 15/17 samples and chromosome 3p and 8q copy numbers matched the primary tumour in 19/21 and 18/20 samples, respectively. Collectively, a clinically-relevant molecular classification of the primary tumour could be inferred from 29/65 vitreous fluids. CONCLUSIONS This proof-of-concept study shows that substantial amounts of DNA could be detected in vitreous fluids from uveal melanoma patients, including melanoma-cell derived DNA in 69% of the samples. Prognostically-relevant genetic alterations of the primary tumour could be identified in 45% of the patients. A follow-up study is needed to evaluate our approach in a prospective clinical context. Additionally, our work highlights improved possibilities to sensitively analyse scarce and heterogeneous tumour biopsies, with potential application in other malignancies.
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Affiliation(s)
- R J Nell
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
| | - M Versluis
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - N V Menger
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - M C Gelmi
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - T H K Vu
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - R M Verdijk
- Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands
- Department of Pathology, Section Ophthalmic Pathology, Erasmus MC University Medical Center, Rotterdam, the Netherlands
| | - G P M Luyten
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - M J Jager
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - P A van der Velden
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
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Musa M, Zhu Z, Takahashi H, Shinoda W, Baba Y, Yasui T. Selective adsorption of unmethylated DNA on ZnO nanowires for separation of methylated DNA. LAB ON A CHIP 2025; 25:1637-1646. [PMID: 39792009 DOI: 10.1039/d4lc00893f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2025]
Abstract
DNA methylation is a crucial epigenetic modification used as a biomarker for early cancer progression. However, existing methods for DNA methylation analysis are complex, time-consuming, and prone to DNA degradation. This work demonstrates selective capture of unmethylated DNAs using ZnO nanowires without chemical or biological modifications, thereby concentrating methylated DNA, particularly those with high methylation levels that can predict cancer risk. We observe varying affinities between methylated and unmethylated DNA on ZnO nanowires, which may be influenced by differences in hydrogen bonding strength, potentially related to the effects of methylation on DNA strand behavior, including self-aggregation and stretching inhibition. As a result, the nanowire-based microfluidic device effectively collects unmethylated DNA, leading to a significantly increased ratio of methylated to unmethylated DNA, particularly for collecting low-concentration methylated DNA. This simplified microfluidic device, composed of ZnO nanowires, enables direct separation of specific methylated DNA, offering a potential approach for DNA methylation mapping in clinical disease diagnostics.
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Affiliation(s)
- Marina Musa
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
| | - Zetao Zhu
- Department of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan.
| | - Hiromi Takahashi
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
| | - Wataru Shinoda
- Research Institute for Interdisciplinary Science, Okayama University, Okayama, 700-8530, Japan
| | - Yoshinobu Baba
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
- Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
- Institute of Quantum Life Science, National Institutes for Quantum Science and Technology (QST), Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan
| | - Takao Yasui
- Department of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan.
- Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
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Gezer U, Özgür E, Yörüker EE, Polatoglou E, Holdenrieder S, Bronkhorst A. LINE-1 cfDNA Methylation as an Emerging Biomarker in Solid Cancers. Cancers (Basel) 2024; 16:3725. [PMID: 39594682 PMCID: PMC11592170 DOI: 10.3390/cancers16223725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Revised: 11/01/2024] [Accepted: 11/03/2024] [Indexed: 11/28/2024] Open
Abstract
Epigenetic dysregulation is a hallmark of many human malignancies, with DNA methylation being a primary mechanism influencing gene expression and maintaining genomic stability. Genome-wide hypomethylation, characteristic of many cancers, is partly attributed to the demethylation of repetitive elements, including LINE-1, a prevalent non-LTR retrotransposon. The methylation status of LINE-1 is closely associated with overall genomic methylation levels in tumors. cfDNA comprises extracellular DNA fragments found in bodily fluids such as plasma, serum, and urine, offering a dynamic snapshot of the genetic and epigenetic landscape of tumors. This real-time sampling provides a minimally invasive avenue for cancer diagnostics, prognostics, and monitoring. The methylation status of LINE-1 in cfDNA has emerged as a promising biomarker, with several studies highlighting its potential in diagnosing and predicting outcomes in cancer patients. Recent research also suggests that cfDNA-based LINE-1 methylation analysis could serve as a valuable tool in evaluating the efficacy of cancer therapies, including immunotherapy. The growing clinical significance of cfDNA calls for a closer examination of its components, particularly repetitive elements like LINE-1. Despite their importance, the role of LINE-1 elements in cfDNA has not been thoroughly gauged. We aim to address this gap by reviewing the current literature on LINE-1 cfDNA assays, focusing on their potential applications in diagnostics and disease monitoring.
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Affiliation(s)
- Ugur Gezer
- Department of Basic Oncology, Oncology Institute, Istanbul University, 34093 Istanbul, Türkiye; (U.G.); (E.Ö.); (E.E.Y.)
| | - Emre Özgür
- Department of Basic Oncology, Oncology Institute, Istanbul University, 34093 Istanbul, Türkiye; (U.G.); (E.Ö.); (E.E.Y.)
| | - Ebru E. Yörüker
- Department of Basic Oncology, Oncology Institute, Istanbul University, 34093 Istanbul, Türkiye; (U.G.); (E.Ö.); (E.E.Y.)
| | - Eleni Polatoglou
- Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center, Technical University Munich, 80636 Munich, Germany (S.H.)
| | - Stefan Holdenrieder
- Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center, Technical University Munich, 80636 Munich, Germany (S.H.)
| | - Abel Bronkhorst
- Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center, Technical University Munich, 80636 Munich, Germany (S.H.)
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Trentin L, Basile D, Lazzari E, Fietta E, Rossi A, Graziani F, Cappetta A, Simionato F, D'Amore E, Perbellini O, Aprile G. Implementation of a MSRE ddPCR method for the detection of methylated WIF1 and NPY genes in colorectal cancer patients. TUMORI JOURNAL 2024; 110:375-385. [PMID: 39101541 DOI: 10.1177/03008916241261675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/06/2024]
Abstract
BACKGROUND Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite. METHODS AND RESULTS We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion. CONCLUSIONS We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.
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Affiliation(s)
- Luca Trentin
- AULSS 8 Berica Ospedale San Bortolo, Laboratory of Haematology, Vicenza, Italy
- AULSS 2 Marca Trevigiana, Histocompatibility Laboratory, Treviso, Italy
| | - Debora Basile
- AULSS 8 Berica Ospedale San Bortolo, UOC Oncologia, Vicenza, Italy
- Unit of Medical Oncology, Ospedale San Giovanni di Dio, Crotone, Italy
| | - Elena Lazzari
- AULSS 8 Berica Ospedale San Bortolo, UOC Anatomia Patologica, Vicenza, Italy
| | - Elena Fietta
- AULSS 8 Berica Ospedale San Bortolo, Laboratory of Haematology, Vicenza, Italy
| | - Alice Rossi
- AULSS 8 Berica Ospedale San Bortolo, UOC Oncologia, Vicenza, Italy
| | | | | | | | - Emanuele D'Amore
- AULSS 8 Berica Ospedale San Bortolo, UOC Anatomia Patologica, Vicenza, Italy
| | - Omar Perbellini
- AULSS 8 Berica Ospedale San Bortolo, Laboratory of Haematology, Vicenza, Italy
| | - Giuseppe Aprile
- AULSS 8 Berica Ospedale San Bortolo, UOC Oncologia, Vicenza, Italy
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Gattuso G, Lavoro A, Caltabiano R, Madonna G, Capone M, Ascierto PA, Falzone L, Libra M, Candido S. Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots. Int J Mol Med 2024; 53:42. [PMID: 38488030 PMCID: PMC10998716 DOI: 10.3892/ijmm.2024.5366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 11/23/2023] [Indexed: 03/19/2024] Open
Abstract
DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high‑throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE‑ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.
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Affiliation(s)
- Giuseppe Gattuso
- Department of Biomedical and Biotechnological Sciences, University of Catania, I‑95123 Catania, Italy
| | - Alessandro Lavoro
- Department of Biomedical and Biotechnological Sciences, University of Catania, I‑95123 Catania, Italy
| | - Rosario Caltabiano
- Department of Medical and Surgical Sciences and Advanced Technologies 'G.F. Ingrassia', University of Catania, I‑95123 Catania, Italy
| | - Gabriele Madonna
- Melanoma Cancer Immunotherapy and Innovative Therapy Unit, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, I‑80131 Naples, Italy
| | - Mariaelena Capone
- Melanoma Cancer Immunotherapy and Innovative Therapy Unit, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, I‑80131 Naples, Italy
| | - Paolo Antonio Ascierto
- Melanoma Cancer Immunotherapy and Innovative Therapy Unit, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, I‑80131 Naples, Italy
| | - Luca Falzone
- Epidemiology and Biostatistics Unit, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, I‑80131 Naples, Italy
| | - Massimo Libra
- Department of Biomedical and Biotechnological Sciences, University of Catania, I‑95123 Catania, Italy
| | - Saverio Candido
- Department of Biomedical and Biotechnological Sciences, University of Catania, I‑95123 Catania, Italy
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7
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Zhang H, Liu L, Li M. Mini-review of DNA Methylation Detection Techniques and Their Potential Applications in Disease Diagnosis, Prognosis, and Treatment. ACS Sens 2024; 9:1089-1103. [PMID: 38365574 DOI: 10.1021/acssensors.3c02328] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/18/2024]
Abstract
DNA methylation is the dominant epigenetic mechanism for regulating gene expression in mammals, playing crucial roles in development, differentiation, and tissue homeostasis. Aberrations in DNA methylation are closely associated with the potential onset of various diseases. Consequently, numerous DNA methylation detection techniques have been successively developed. These methods not only facilitate the exploration of disease mechanisms but also hold significant promise for the development of diagnostic and prognostic strategies. In this Perspective, we present a comprehensive overview of commonly employed DNA methylation detection techniques as well as biosensing based on their underlying analytical techniques. For its medical applications, we begin by examining the pathogenesis of different diseases and then proceed to discuss how relevant technologies are applied in the context of these specific medical conditions. Additionally, we briefly discuss the current limitations of these techniques and highlight future challenges in advancing methylation detection and analysis methodologies.
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Affiliation(s)
- Huaming Zhang
- Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212013, China
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
| | - Lei Liu
- Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang 212013, China
| | - Min Li
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
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8
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Aleyasin SA, Moradi A, Abolhasani N, Abdollahi M. Investigating FGFR2 gene as a blood-based epigenetic biomarker in gastric cancer. Mol Biol Rep 2024; 51:253. [PMID: 38302798 DOI: 10.1007/s11033-023-09082-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Accepted: 11/30/2023] [Indexed: 02/03/2024]
Abstract
BACKGROUND Gastric adenocarcinoma is a prevalent form of cancer that often remains undetected in its early stages due to the lack of specific symptoms. This delayed diagnosis leads to poor clinical outcomes, underscoring the need for an effective and non-invasive method for early detection. Recent advances in cancer epigenetics have led to the identification of biomarkers that have the potential to revolutionize the early detection and monitoring of this disease. One such promising biomarker is the methylation of the FGFR2 promoter. This study aims to measure the methylation levels of a specific CpG site in the FGFR2 promoter gene in DNA extracted from blood leukocytes from patients with intestinal metaplasia, gastric cancer, and healthy control. MATERIAL AND METHODS The CpG site of the FGFR2 gene promoter was identified in its control region. Methylation alteration of the selected FGFR2 CpG site was determined through the (methylation-sensitive restriction enzyme) MSRE-qPCR. Genomic DNA was extracted from one hundred twenty-five participants. RESULTS The normal group had mean methylation levels of 93.23 ± 4.929%, while the IM group had a level of 69.85 ± 27.15%. In GC patients, the levels varied, with 25.96 ± 18.98% in the intestinal type and 28.30 ± 16.07% in the diffuse type. The methylation levels in the IM and GC patients were significantly lower than those in the normal control group. However, no significant difference was observed between the methylation status of the intestinal type of GC and the diffuse type. The Receiver operating characteristic (ROC) curve analysis showed that FGFR2 CpG methylation levels in GC patients compared to normal controls had a high sensitivity of 100% and specificity of 100%, with a cut-off of < 74.25%; when GC patients were compared to IM patients, the sensitivity was 85%, and the specificity was 80%, with a cut-off < 44.45%. CONCLUSIONS The potential of the FGFR2 methylation status as a non-invasive biomarker lies in its ability to be detected in blood leukocytes, which makes it a promising tool for the early detection of intestinal metaplasia and gastric cancer. This could significantly improve the detection and management of these gastric conditions.
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Affiliation(s)
- Seyed Ahmad Aleyasin
- Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 17 Km Tehran-Karaj Highway, Pajoohesh Blvd, Tehran, Iran.
| | - Arash Moradi
- Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 17 Km Tehran-Karaj Highway, Pajoohesh Blvd, Tehran, Iran
| | - Naeimeh Abolhasani
- Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 17 Km Tehran-Karaj Highway, Pajoohesh Blvd, Tehran, Iran
| | - Mahvash Abdollahi
- Department of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 17 Km Tehran-Karaj Highway, Pajoohesh Blvd, Tehran, Iran
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Kolkman R, Michel-Souzy S, Wasserberg D, Segerink LI, Huskens J. Density Control over MBD2 Receptor-Coated Surfaces Provides Superselective Binding of Hypermethylated DNA. ACS APPLIED MATERIALS & INTERFACES 2022; 14:40579-40589. [PMID: 36052432 PMCID: PMC9478954 DOI: 10.1021/acsami.2c09641] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 08/26/2022] [Indexed: 06/15/2023]
Abstract
Using the biomarker hypermethylated DNA (hmDNA) for cancer detection requires a pretreatment to isolate or concentrate hmDNA from nonmethylated DNA. Affinity chromatography using a methyl binding domain-2 (MBD2) protein can be used, but the relatively low enrichment selectivity of MBD2 limits its clinical applicability. Here, we developed a superselective, multivalent, MBD2-coated platform to improve the selectivity of hmDNA enrichment. The multivalent platform employs control over the MBD2 surface receptor density, which is shown to strongly affect the binding of DNA with varying degrees of methylation, improving both the selectivity and the affinity of DNAs with higher numbers of methylation sites. Histidine-10-tagged MBD2 was immobilized on gold surfaces with receptor density control by tuning the amount of nickel nitrilotriacetic acid (NiNTA)-functionalized thiols in a thiol-based self-assembled monolayer. The required MBD2 surface receptor densities for DNA surface binding decreases for DNA with higher degrees of methylation. Both higher degrees of superselectivity and surface coverages were observed upon DNA binding at increasing methylation levels. Adopting the findings of this study into hmDNA enrichment of clinical samples has the potential to become more selective and sensitive than current MBD2-based methods and, therefore, to improve cancer diagnostics.
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Affiliation(s)
- Ruben
W. Kolkman
- Molecular
Nanofabrication Group, Department for Molecules & Materials, MESA+
Institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
- BIOS
Lab on a Chip Group, MESA+ Institute and TechMed Centre, Max Planck
Institute for Complex Fluid Dynamics, Faculty of Electrical Engineering,
Mathematics and Computer Science, University
of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
| | - Sandra Michel-Souzy
- Biomolecular
Nanotechnology Group, Department for Molecules & Materials, MESA+
Institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
| | - Dorothee Wasserberg
- BIOS
Lab on a Chip Group, MESA+ Institute and TechMed Centre, Max Planck
Institute for Complex Fluid Dynamics, Faculty of Electrical Engineering,
Mathematics and Computer Science, University
of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
| | - Loes I. Segerink
- BIOS
Lab on a Chip Group, MESA+ Institute and TechMed Centre, Max Planck
Institute for Complex Fluid Dynamics, Faculty of Electrical Engineering,
Mathematics and Computer Science, University
of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
| | - Jurriaan Huskens
- Molecular
Nanofabrication Group, Department for Molecules & Materials, MESA+
Institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
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10
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Li J, Li H, Run ZC, Wang ZL, Jiang T, An Y, Li Z. RASSF1A methylation as a biomarker for detection of colorectal cancer and hepatocellular carcinoma. World J Gastrointest Oncol 2022; 14:1574-1584. [PMID: 36160746 PMCID: PMC9412931 DOI: 10.4251/wjgo.v14.i8.1574] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Revised: 07/02/2022] [Accepted: 07/22/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Studies have validated the potential of methylated cell-free DNA as a biomarker in various tumors, and methylated DNA in plasma may be a potential biomarker for cancer.
AIM To evaluate the diagnostic value of RASSF1A methylation in plasma for colorectal cancer (CRC) and hepatocellular carcinoma (HCC).
METHODS A total of 92 CRC patients, 67 colorectal polyp (CRP) patients, 63 HCC patients, and 66 liver cirrhosis (LC) patients were enrolled. The plasma DNA was subjected to DNA extraction, double-strand DNA concentration determination, bisulfite conversion, purification, single-strand DNA concentration determination, and digital polymerase chain reaction (PCR) detection. The methylation rate was calculated. The diagnostic value was evaluated by the area under the curve (AUC).
RESULTS The age and sex in the CRC and CRP groups and the HCC and LC groups were also matched. The DNA methylation rate of RASSF1A in plasma in the CRC group was 2.87 ± 1.80, and that in the CRP group was 1.50 ± 0.64. DNA methylation of RASSF1A in plasma showed a significant difference between the CRC and CRP groups. The AUC of RASSF1A methylation for discriminating the CRC and CRP groups was 0.82 (0.76-0.88). The AUCs of T1, T2, T3 and T4 CRC and CRP were 0.83 (0.72-0.95), 0.87 (0.78-0.95), 0.86 (0.77-0.95), and 0.75 (0.64-0.85), respectively. The DNA methylation rate of RASSF1A in plasma in the HCC group was 4.45 ± 2.93, and that in the LC group was 2.46 ± 2.07. DNA methylation of RASSF1A in plasma for the HCC and LC groups showed a significant difference. The AUC of RASSF1A methylation for discriminating the HCC and LC groups was 0.70 (0.60-0.79). The AUCs of T1, T2, T3 and T4 HCC and LC were 0.80 (0.61, 1.00), 0.74 (0.59-0.88), 0.60 (0.42-0.79), and 0.68 (0.53-0.82), respectively.
CONCLUSION RASSF1A methylation in plasma detected by digital PCR may be a potential biomarker for CRC and HCC.
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Affiliation(s)
- Jian Li
- Department of General Surgery, Affiliated Tumor Hospital of Zhengzhou University, Henan Tumor Hospital, Zhengzhou 450000, Henan Province, China
| | - Huan Li
- Department of Gastroenterology, Sixth Medical Center of Chinese PLA General Hospital, Beijing 100048, China
| | - Zeng-Ci Run
- Department of General Surgery, Affiliated Tumor Hospital of Zhengzhou University, Henan Tumor Hospital, Zhengzhou 450000, Henan Province, China
| | - Zhen-Lei Wang
- Department of General Surgery, Affiliated Tumor Hospital of Zhengzhou University, Henan Tumor Hospital, Zhengzhou 450000, Henan Province, China
| | - Tao Jiang
- Medicine Innovation Research Division of Chinese PLA General Hospital, Beijing 100853, China
| | - Yang An
- Faculty of Hepato-Pancreato-Biliary Surgery, Sixth Medical Center of Chinese PLA General Hospital, Beijing 100048, China
| | - Zhi Li
- Department of General Surgery, Affiliated Tumor Hospital of Zhengzhou University, Henan Tumor Hospital, Zhengzhou 450000, Henan Province, China
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11
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A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation. Anal Chim Acta 2022; 1212:339914. [DOI: 10.1016/j.aca.2022.339914] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 05/03/2022] [Accepted: 05/04/2022] [Indexed: 12/26/2022]
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12
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van de Leemkolk FEM, Nell RJ, Versluis M, de Koning EJP, Huurman VAL, Alwayn IPJ, Ploeg RJ, van der Velden PA, Engelse MA. Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction. Transpl Int 2022; 35:10167. [PMID: 35462792 PMCID: PMC9022224 DOI: 10.3389/ti.2022.10167] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Accepted: 03/15/2022] [Indexed: 11/13/2022]
Abstract
Assessment of specific β-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to β-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of β-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as a reflection of the relative amount of lysed β-cells.
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Affiliation(s)
- Fenna E. M. van de Leemkolk
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Department of Surgery, Leiden University Medical Center, Leiden, Netherlands
- *Correspondence: Fenna E. M. van de Leemkolk, ; Marten A. Engelse,
| | - Rogier J. Nell
- Department of Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
| | - Mieke Versluis
- Department of Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
| | - Eelco J. P. de Koning
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Department of Internal Medicine, Leiden University Medical Center, Leiden, Netherlands
| | - Volkert A. L. Huurman
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Department of Surgery, Leiden University Medical Center, Leiden, Netherlands
| | - Ian P. J. Alwayn
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Department of Surgery, Leiden University Medical Center, Leiden, Netherlands
| | - Rutger J. Ploeg
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom
| | | | - Marten A. Engelse
- LUMC Transplant Center, Leiden University Medical Center, Leiden, Netherlands
- Department of Internal Medicine, Leiden University Medical Center, Leiden, Netherlands
- *Correspondence: Fenna E. M. van de Leemkolk, ; Marten A. Engelse,
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13
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Nell RJ, Zoutman WH, Calbet-Llopart N, Garcia AP, Menger NV, Versluis M, Puig S, Gruis NA, van der Velden PA. Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR. J Mol Diagn 2021; 24:88-100. [PMID: 34775028 DOI: 10.1016/j.jmoldx.2021.10.007] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Revised: 10/06/2021] [Accepted: 10/13/2021] [Indexed: 11/25/2022] Open
Abstract
An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as copy number instability can disturb the underlying mathematical model. For example, approximately 24% of the tumors from The Cancer Genome Atlas pan-cancer data set carried a copy number alteration affecting our TRB gene T-cell marker, which would cause an underestimation or overestimation of the T-cell fraction. In this study, we introduce a multiplex digital PCR experimental setup to quantify T cells in copy number unstable DNA samples. By implementing a so-called regional corrector, genetic alterations involving the T-cell marker locus can be recognized and corrected for. This novel setup is evaluated mathematically in silico and validated in vitro by measuring T-cell presence in various samples with a known T-cell fraction. The utility of the approach is further demonstrated in copy number altered cutaneous melanomas. Our novel multiplex setup provides a simple, but accurate, DNA-based T-cell quantification in both copy number stable and unstable specimens. This approach has potential clinical and diagnostic applications, as it does not depend on availability of T-cell epitopes, has low requirements for sample quantity and quality, and can be performed in a relatively easy experiment.
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Affiliation(s)
- Rogier J Nell
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - Willem H Zoutman
- Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands
| | - Neus Calbet-Llopart
- Department of Dermatology, Hospital Clínic de Barcelona, IDIBAPS, University of Barcelona, Centro Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Spain
| | - Adriana P Garcia
- Department of Pathology, Hospital Clínic de Barcelona, Barcelona, Spain
| | - Nino V Menger
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - Mieke Versluis
- Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
| | - Susana Puig
- Department of Dermatology, Hospital Clínic de Barcelona, IDIBAPS, University of Barcelona, Centro Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Spain
| | - Nelleke A Gruis
- Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands
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14
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Nell RJ, van Steenderen D, Menger NV, Weitering TJ, Versluis M, van der Velden PA. Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR. Hum Mutat 2020; 41:2205-2216. [PMID: 32906203 PMCID: PMC7756443 DOI: 10.1002/humu.24111] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 08/22/2020] [Accepted: 09/06/2020] [Indexed: 12/20/2022]
Abstract
Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation‐sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context‐density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.
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Affiliation(s)
- Rogier J Nell
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
| | - Debby van Steenderen
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
| | - Nino V Menger
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
| | - Thomas J Weitering
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
| | - Mieke Versluis
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
| | - Pieter A van der Velden
- Department of Ophthalmology, Leiden University Medical Center, Leiden, South Holland, The Netherlands
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