Circulating tumor DNA methylation detection as biomarker and its application in tumor liquid biopsy: advances and challenges

Abstract

Circulating tumor DNA (ctDNA) methylation, an innovative liquid biopsy biomarker, has emerged as a promising tool in early cancer diagnosis, monitoring, and prognosis prediction. As a noninvasive approach, liquid biopsy overcomes the limitations of traditional tissue biopsy. Among various biomarkers, ctDNA methylation has garnered significant attention due to its high specificity and early detection capability across diverse cancer types. Despite its immense potential, the clinical application of ctDNA methylation faces substantial challenges pertaining to sensitivity, specificity, and standardization. In this review, we begin by introducing the basic biology and common detection techniques of ctDNA methylation. We then explore recent advancements and the challenges faced in the clinical application of ctDNA methylation in liquid biopsies. This includes progress in early screening and diagnosis, identification of clinical molecular subtypes, monitoring of recurrence and minimal residual disease (MRD), prediction of treatment response and prognosis, assessment of tumor burden, and determination of tissue origin. Finally, we discuss the future perspectives and challenges of ctDNA methylation detection in clinical applications. This comprehensive overview underscores the vital role of ctDNA methylation in enhancing cancer diagnostic accuracy, personalizing treatments, and effectively monitoring disease progression, providing valuable insights for future research and clinical practice.

Keywords

biomarker / circulating tumor DNA / liquid biopsy / methylation / tumor

References

[1] Luo H, Wei W, Ye Z, Zheng J, Xu RH. Liquid biopsy of methylation biomarkers in cell-free DNA. Trends Mol Med. 2021; 27(5): 482-500.

[2] Shields MD, Chen K, Dutcher G, Patel I, Pellini B. Making the rounds: exploring the role of circulating tumor DNA (ctDNA) in non-small cell lung cancer. Int J Mol Sci. 2022; 23(16): 9006.

[3] Zhu L, Sun HT, Wang S, et al. Isolation and characterization of exosomes for cancer research. J Hematol Oncol. 2020; 13(1): 152.

[4] Mishra V, Singh A, Chen X, et al. Application of liquid biopsy as multi-functional biomarkers in head and neck cancer. Br J Cancer. 2022; 126(3): 361-370.

[5] Soda N, Rehm BHA, Sonar P, Nguyen NT, Shiddiky MJA. Advanced liquid biopsy technologies for circulating biomarker detection. J Mater Chem B. 2019; 7(43): 6670-6704.

[6] Li Y, Fan Z, Meng Y, Liu S, Zhan H. Blood-based DNA methylation signatures in cancer: a systematic review. Biochim Biophys Acta Mol Basis Dis. 2023; 1869(1): 166583.

[7] Warton K, Lin V, Navin T, et al. Methylation-capture and next-generation sequencing of free circulating DNA from human plasma. BMC Genomics. 2014; 15(1): 476.

[8] Tserpeli V, Stergiopoulou D, Londra D, et al. Prognostic significance of SLFN11 methylation in plasma cell-free DNA in advanced high-grade serous ovarian cancer. Cancers (Basel). 2021; 14(1): 4.

[9] Alix-Panabieres C, Pantel K. Challenges in circulating tumour cell research. Nat Rev Cancer. 2014; 14(9): 623-631.

[10] Welch HG, Robertson DJ. Colorectal cancer on the decline–why screening can’t explain it all. N Engl J Med. 2016; 374(17): 1605-1607.

[11] Yamada T, Matsuda A, Koizumi M, et al. Liquid biopsy for the management of patients with colorectal cancer. Digestion. 2019; 99(1): 39-45.

[12] Liu H, Ye D, Chen A, et al. A pilot study of new promising non-coding RNA diagnostic biomarkers for early-stage colorectal cancers. Clin Chem Lab Med. 2019; 57(7): 1073-1083.

[13] Shitara K, Muro K, Watanabe J, et al. Baseline ctDNA gene alterations as a biomarker of survival afterpanitumumab and chemotherapy in metastatic colorectal cancer. Nat Med. 2024; 30: 730-739. Published online February 12, 2024.

[14] Li Y, Xu J, Chen C, et al. Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection. Genome Med. 2024; 16(1): 9.

[15] Mattox AK, Douville C, Wang Y, et al. The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer. Cancer Discov. 2023; 13(10): 2166-2179.

[16] Danese E, Montagnana M, Lippi G. Circulating molecular biomarkers for screening or early diagnosis of colorectal cancer: which is ready for prime time?. Ann Transl Med. 2019; 7(21): 610.

[17] Mali SB. Molecular screening of head neck cancer. Oral Oncol. 2023; 144: 106481.

[18] Parikh AR, Van Seventer EE, Siravegna G, et al. Minimal residual disease detection using a plasma-only circulating tumor DNA assay in patients with colorectal cancer. Clin Cancer Res. 2021; 27(20): 5586-5594.

[19] Baca SC, Seo JH, Davidsohn MP, et al. Liquid biopsy epigenomic profiling for cancer subtyping. Nat Med. 2023; 29(11): 2737-2741.

[20] Nassar FJ, Msheik ZS, Nasr RR, Temraz SN. Methylated circulating tumor DNA as a biomarker for colorectal cancer diagnosis, prognosis, and prediction. Clin Epigenetics. 2021; 13(1): 111.

[21] Lin J, Ma L, Zhang D, et al. Tumour biomarkers-Tracing the molecular function and clinical implication. Cell Prolif. 2019; 52(3): e12589.

[22] Weisenberger DJ. Characterizing DNA methylation alterations from The Cancer Genome Atlas. J Clin Invest. 2014; 124(1): 17-23.

[23] Hu X, Estecio MR, Chen R, et al. Evolution of DNA methylome from precancerous lesions to invasive lung adenocarcinomas. Nat Commun. 2021; 12(1): 687.

[24] Wu X, Li J, Gassa A, et al. Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma. Int J Biol Sci. 2020; 16(9): 1551-1562.

[25] Wang W, Zhu X, Zhang X, et al. Recurrence risk assessment for stage III colorectal cancer based on five methylation biomarkers in plasma cell-free DNA. J Pathol. 2023; 259(4): 376-387.

[26] Tian XP, Zhang YC, Lin NJ, et al. Diagnostic performance and prognostic value of circulating tumor DNA methylation marker in extranodal natural killer/T cell lymphoma. Cell Rep Med. 2023; 4(2): 100859.

[27] Cao M, Shi J, Xia C, et al. Efficacy of ctDNA methylation combined with traditional detection modality to detect liver cancer among high-risk patients: a multicenter diagnostic trial. Chin J Cancer Res. 2023; 35(1): 58-65.

[28] Warton K, Mahon KL, Samimi G. Methylated circulating tumor DNA in blood: power in cancer prognosis and response. Endocr Relat Cancer. 2016; 23(3): R157-R171.

[29] Lo YMD, Han DSC, Jiang P, Chiu RWK. Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies. Science. 2021; 372(6538): eaaw3616.

[30] Chemi F, Pearce SP, Clipson A, et al. cfDNA methylome profiling for detection and subtyping of small cell lung cancers. Nat Cancer. 2022; 3(10): 1260-1270.

[31] Heeke S, Gay CM, Estecio MR, et al. Tumor-and circulating-free DNA methylation identifies clinically relevant small cell lung cancer subtypes. Cancer Cell. 2024; 42(2): 225-237.

[32] Gay CM, Stewart CA, Park EM, et al. Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities. Cancer Cell. 2021; 39(3): 346-360.

[33] Xie M, Chugh P, Broadhurst H, et al. Abstract CT024: durvalumab (D) + platinum-etoposide (EP) in 1L extensive-stage small-cell lung cancer (ES-SCLC): exploratory analysis of SCLC molecular subtypes in CASPIAN. Cancer Res. 2022; 82: CT024.

[34] Liang WW, Lu RJ, Jayasinghe RG, et al. Integrative multi-omic cancer profiling reveals DNA methylation patterns associated with therapeutic vulnerability and cell-of-origin. Cancer Cell. 2023; 41(9): 1567-1585.

[35] Wang T, Li P, Qi Q, et al. A multiplex blood-based assay targeting DNA methylation in PBMCs enables early detection of breast cancer. Nat Commun. 2023; 14(1): 4724.

[36] Xie Y, Li P, Sun D, et al. DNA methylation-based testing in peripheral blood mononuclear cells enables accurate and early detection of colorectal cancer. Cancer Res. 2023; 83(21): 3636-3649.

[37] Ul Haq S, Schmid S, Aparnathi MK, et al. Cell-free DNA methylation-defined prognostic subgroups in small-cell lung cancer identified by leukocyte methylation subtraction. iScience. 2022; 25(12): 105487.

[38] Bent A, Kopetz S. Going with the flow. The promise of plasma-only circulating tumor DNA assays. Clin Cancer Res. 2021; 27(20): 5449-5451.

[39] Taieb J, Taly V, Henriques J, et al. Prognostic value and relation with adjuvant treatment duration of ctDNA in stage III colon cancer: a post hoc analysis of the PRODIGE-GERCOR IDEA-France trial. Clin Cancer Res. 2021; 27(20): 5638-5646.

[40] Thomsen CB, Andersen RF, Steffensen KD, Adimi P, Jakobsen A. Delta tocotrienol in recurrent ovarian cancer. A phase II trial. Pharmacol Res. 2019; 141: 392-396.

[41] Delmonico L, Alves G, Bines J. Cell free DNA biology and its involvement in breast carcinogenesis. Adv Clin Chem. 2020; 97: 171-223.

[42] Li J, Zhao S, Lee M, et al. Reliable tumor detection by whole-genome methylation sequencing of cell-free DNA in cerebrospinal fluid of pediatric medulloblastoma. Sci Adv. 2020; 6(42): eabb5427.

[43] Murray DH, Symonds EL, Young GP, et al. Relationship between post-surgery detection of methylated circulating tumor DNA with risk of residual disease and recurrence-free survival. J Cancer Res Clin Oncol. 2018; 144(9): 1741-1750.

[44] Leygo C, Williams M, Jin HC, et al. DNA methylation as a noninvasive epigenetic biomarker for the detection of cancer. Dis Markers. 2017; 2017: 3726595.

[45] Yizhak K, Aguet F, Kim J, et al. RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Science. 2019; 364(6444): eaaw0726.

[46] Guo S, Diep D, Plongthongkum N, Fung HL, Zhang K, Zhang K. Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA. Nat Genet. 2017; 49(4): 635-642.

[47] Musher BL, Melson JE, Amato G, et al. Evaluation of circulating tumor DNA for methylated BCAT1 and IKZF1 to detect recurrence of stage II/Stage III colorectal cancer (CRC). Cancer Epidemiol Biomarkers Prev. 2020; 29(12): 2702-2709.

[48] Zhao F, Bai P, Xu J, et al. Efficacy of cell-free DNA methylation-based blood test for colorectal cancer screening in high-risk population: a prospective cohort study. Mol Cancer. 2023; 22(1): 157.

[49] van der Pol Y, Moldovan N, Ramaker J, et al. The landscape of cell-free mitochondrial DNA in liquid biopsy for cancer detection. Genome Biol. 2023; 24(1): 229.

[50] Gao Y, Zhao H, An K, et al. Whole-genome bisulfite sequencing analysis of circulating tumour DNA for the detection and molecular classification of cancer. Clin Transl Med. 2022; 12(8): e1014.

[51] Gorgannezhad L, Umer M, Islam MN, Nguyen NT, Shiddiky MJA. Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies. Lab Chip. 2018; 18(8): 1174-1196.

[52] Li L, Fu K, Zhou W, Snyder M. Applying circulating tumor DNA methylation in the diagnosis of lung cancer. Precis Clin Med. 2019; 2(1): 45-56.

[53] Sun Z, Cunningham J, Slager S, Kocher JP. Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis. Epigenomics. 2015; 7(5): 813-828.

[54] Wen L, Li J, Guo H, et al. Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients. Cell Res. 2015; 25(11): 1250-1264.

[55] Shen X, Xu M, Wang H, et al. Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification. Biosens Bioelectron. 2023; 237: 115550.

[56] Wu H, Wu X, Shen L, Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol. 2014; 32(12): 1231-1240.

[57] Liang N, Li B, Jia Z, et al. Ultrasensitive detection of circulating tumour DNA via deep methylation sequencing aided by machine learning. Nat Biomed Eng. 2021; 5(6): 586-599.

[58] Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV, Consortium C. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol. 2020; 31(6): 745-759.

[59] Huang Y, Pastor WA, Zepeda-Martinez JA, Rao A. The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc. 2012; 7(10): 1897-1908.

[60] Calapre L, Giardina T, Robinson C, et al. Locus-specific concordance of genomic alterations between tissue and plasma circulating tumor DNA in metastatic melanoma. Mol Oncol. 2019; 13(2): 171-184.

[61] Maggi EC, Gravina S, Cheng H, et al. Development of a method to implement whole-genome bisulfite sequencing of cfDNA from cancer patients and a mouse tumor model. Front Genet. 2018; 9: 6.

[62] Geertsen L, Koldby KM, Thomassen M, Kruse T, Lund L. Circulating tumor DNA in patients with renal cell carcinoma. a systematic review of the literature. Eur Urol Open Sci. 2022; 37: 27-35.

[63] Tzanikou E, Lianidou E. The potential of ctDNA analysis in breast cancer. Crit Rev Clin Lab Sci. 2020; 57(1): 54-72.

[64] Chen K, Kang G, Zhao H, et al. Liquid biopsy in newly diagnosed patients with locoregional (I-IIIA) non-small cell lung cancer. Expert Rev Mol Diagn. 2019; 19(5): 419-427.

[65] Nuzzo PV, Berchuck JE, Korthauer K, et al. Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med. 2020; 26(7): 1041-1043.

[66] Gouda MA, Duose DY, Lapin M, et al. Mutation-agnostic detection of colorectal cancer using liquid biopsy-based methylation-specific signatures. Oncologist. 2023; 28(4): 368-372.

[67] Mastoraki S, Strati A, Tzanikou E, et al. ESR1 methylation: a liquid biopsy-based epigenetic assay for the follow-up of patients with metastatic breast cancer receiving endocrine treatment. Clin Cancer Res. 2018; 24(6): 1500-1510.

[68] Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM). a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 2007; 35(6): e41.

[69] Giannopoulou L, Chebouti I, Pavlakis K, Kasimir-Bauer S, Lianidou ES. RASSF1A promoter methylation in high-grade serous ovarian cancer: a direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA. Oncotarget. 2017; 8(13): 21429-21443.

[70] Shan M, Yin H, Li J, et al. Detection of aberrant methylation of a six-gene panel in serum DNA for diagnosis of breast cancer. Oncotarget. 2016; 7(14): 18485-18494.

[71] Yamamoto N, Nakayama T, Kajita M, et al. Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARbeta2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay. Breast Cancer Res Treat. 2012; 132(1): 165-173.

[72] Parisi C, Mastoraki S, Markou A, et al. Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis. Clin Chim Acta. 2016; 461: 156-164.

[73] Yang F, Tang J, Zhao Z, Zhao C, Xiang Y. Circulating tumor DNA. a noninvasive biomarker for tracking ovarian cancer. Reprod Biol Endocrinol. 2021; 19(1): 178.

[74] Aulakh SS, Silverman DA, Young K, Dennis SK, Birkeland AC. The promise of circulating tumor DNA in head and neck cancer. Cancers (Basel). 2022; 14(12): 2968.

[75] Huerta M, Rosello S, Sabater L, et al. Circulating tumor DNA detection by digital-droplet PCR in pancreatic ductal adenocarcinoma: a systematic review. Cancers (Basel). 2021; 13(5): 994.

[76] Taylor SC, Laperriere G, Germain H. Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep. 2017; 7(1): 2409.

[77] Ogaard N, Reinert T, Henriksen TV, et al. Tumour-agnostic circulating tumour DNA analysis for improved recurrence surveillance after resection of colorectal liver metastases: a prospective cohort study. Eur J Cancer. 2022; 163: 163-176.

[78] Zhao Y, Xue F, Sun J, et al. Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma. Clin Epigenetics. 2014; 6(1): 30.

[79] Xu W, Lu J, Zhao Q, et al. Genome-wide plasma cell-free DNA methylation profiling identifies potential biomarkers for lung cancer. Dis Markers. 2019; 2019: 4108474.

[80] Serre D, Lee BH, Ting AH. MBD-isolated genome sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome. Nucleic Acids Res. 2010; 38(2): 391-399.

[81] Xin W, Tu S, Yi S, et al. Clinical significance of tumor suppressor genes methylation in circulating tumor DNA of patients with pancreatic cancer. Gene. 2024; 897: 148078.

[82] Jin S, Zhu D, Shao F, et al. Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy. Proc Natl Acad Sci USA. 2021; 118(5): e2017421118.

[83] Rasmussen SL, Krarup HB, Sunesen KG, et al. Hypermethylated DNA, a circulating biomarker for colorectal cancer detection. PLoS One. 2017; 12(7): e0180809.

[84] Ma ZY, Law WL, Ng EKO, et al. Methylated septin 9 and carcinoembryonic antigen for serological diagnosis and monitoring of patients with colorectal cancer after surgery. Sci Rep. 2019; 9(1): 10326.

[85] Xie L, Jiang X, Li Q, et al. Diagnostic value of methylated septin9 for colorectal cancer detection. Front Oncol. 2018; 8: 247.

[86] Li W, Li Q, Kang S, et al. CancerDetector: ultrasensitive and non-invasive cancer detection at the resolution of individual reads using cell-free DNA methylation sequencing data. Nucleic Acids Res. 2018; 46(15): e89.

[87] Asante DB, Calapre L, Ziman M, Meniawy TM, Gray ES. Liquid biopsy in ovarian cancer using circulating tumor DNA and cells: ready for prime time?. Cancer Lett. 2020; 468: 59-71.

[88] Nguyen VTC, Nguyen TH, Doan NNT, et al. Multimodal analysis of methylomics and fragmentomics in plasma cell-free DNA for multi-cancer early detection and localization. Elife. 2023; 12: RP89083.

[89] Martin-Alonso C, Tabrizi S, Xiong K, et al. Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies. Science. 2024; 383(6680): eadf2341.

[90] Misawa K, Imai A, Matsui H, et al. Identification of novel methylation markers in HPV-associated oropharyngeal cancer: genome-wide discovery, tissue verification and validation testing in ctDNA. Oncogene. 2020; 39(24): 4741-4755.

[91] Lin WH, Xiao J, Ye ZY, et al. Circulating tumor DNA methylation marker MYO1-G for diagnosis and monitoring of colorectal cancer. Clin Epigenetics. 2021; 13(1): 232.

[92] Beltran H, Romanel A, Conteduca V, et al. Circulating tumor DNA profile recognizes transformation to castration-resistant neuroendocrine prostate cancer. J Clin Invest. 2020; 130(4): 1653-1668.

[93] Masud MK, Umer M, Hossain MSA, Yamauchi Y, Nguyen NT, Shiddiky MJA. Nanoarchitecture frameworks for electrochemical miRNA detection. Trends Biochem Sci. 2019; 44(5): 433-452.

[94] Masud MK, Na J, Lin TE, et al. Nanostructured mesoporous gold biosensor for microRNA detection at attomolar level. Biosens Bioelectron. 2020; 168: 112429.

[95] Islam MN, Masud MK, Haque MH, et al. RNA biomarkers: diagnostic and prognostic potentials and recent developments of electrochemical biosensors. Small Methods. 2017; 1(7): 1700131.

[96] Duong BTV, Wu L, Green BJ, et al. A liquid biopsy for detecting circulating mesothelial precursor cells: a new biomarker for diagnosis and prognosis in mesothelioma. EBioMedicine. 2020; 61: 103031.

[97] Clack K, Soda N, Kasetsirikul S, Mahmudunnabi RG, Nguyen NT, Shiddiky MJA. Toward personalized nanomedicine: the critical evaluation of micro and nanodevices for cancer biomarker analysis in liquid biopsy. Small. 2023; 19(15): 2205856.

[98] Zhang P, Wen X, Gu F, et al. Methylation profiling of serum DNA from hepatocellular carcinoma patients using an infinium human methylation 450 BeadChip. Hepatol Int. 2013; 7(3): 893-900.

[99] Mathios D, Johansen JS, Cristiano S, et al. Detection and characterization of lung cancer using cell-free DNA fragmentomes. Nat Commun. 2021; 12(1): 5060.

[100] Nassiri F, Chakravarthy A, Feng S, et al. Detection and discrimination of intracranial tumors using plasma cell-free DNA methylomes. Nat Med. 2020; 26(7): 1044-1047.

[101] Bjerre MT, Norgaard M, Larsen OH, et al. Epigenetic analysis of circulating tumor DNA in localized and metastatic prostate cancer: evaluation of clinical biomarker potential. Cells. 2020; 9(6): 1362.

[102] Witjes JA, Morote J, Cornel EB, et al. Performance of the bladder EpiCheck methylation test for patients under surveillance for non-muscle-invasive bladder cancer: results of a multicenter, prospective, blinded clinical trial. Eur Urol Oncol. 2018; 1(4): 307-313.

[103] Shen SY, Burgener JM, Bratman SV, De Carvalho DD. Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA. Nat Protoc. 2019; 14(10): 2749-2780.

[104] Schutsky EK, DeNizio JE, Hu P, et al. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nat Biotechnol. 2018.

[105] Liu Y, Siejka-Zielińska P, Velikova G, et al. Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat Biotechnol. 2019; 37(4): 424-429.

[106] Tse OYO, Jiang P, Cheng SH, et al. Genome-wide detection of cytosine methylation by single molecule real-time sequencing. Proc Natl Acad Sci USA. 2021; 118(5): e2019768118.

[107] Miller DE, Sulovari A, Wang T, et al. Targeted long-read sequencing identifies missing disease-causing variation. Am J Hum Genet. 2021; 108(8): 1436-1449.

[108] Li X, Guo S, Cui Y, et al. NT-seq: a chemical-based sequencing method for genomic methylome profiling. Genome Biol. 2022; 23(1): 122.

[109] Wang T, Fowler JM, Liu L, et al. Direct enzymatic sequencing of 5-methylcytosine at single-base resolution. Nat Chem Biol. 2023; 19(8): 1004-1012.

[110] Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol. 2020; 31(6): 745-759.

[111] Fiala C, Diamandis EP. A multi-cancer detection test: focus on the positive predictive value. Ann Oncol. 2020; 31(9): 1267-1268.

[112] Li J, Wei L, Zhang X, et al. DISMIR: deep learning-based noninvasive cancer detection by integrating DNA sequence and methylation information of individual cell-free DNA reads. Brief Bioinform. 2021; 22(6): bbab250.

[113] Jamshidi A, Liu MC, Klein EA, et al. Evaluation of cell-free DNA approaches for multi-cancer early detection. Cancer Cell. 2022; 40(12): 1537-1549. e1512.

[114] Wang P, Song Q, Ren J, et al. Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection. Sci Transl Med. 2022; 14(672): eabp8704.

[115] Cheishvili D, Wong C, Karim MM, et al. A high-throughput test enables specific detection of hepatocellular carcinoma. Nat Commun. 2023; 14(1): 3306.

[116] Li S, Zeng W, Ni X, et al. Comprehensive tissue deconvolution of cell-free DNA by deep learning for disease diagnosis and monitoring. Proc Natl Acad Sci USA. 2023; 120(28): e2305236120.

[117] Xiao Y, Ju L, Qian K, et al. Non-invasive diagnosis and surveillance of bladder cancer with driver and passenger DNA methylation in a prospective cohort study. Clin Transl Med. 2022; 12(8): e1008.

[118] Baine MK, Hsieh MS, Lai WV, et al. SCLC subtypes defined by ASCL1, NEUROD1, POU2F3, and YAP1: a comprehensive immunohistochemical and histopathologic characterization. J Thorac Oncol. 2020; 15(12): 1823-1835.

[119] Pedersen SK, Symonds EL, Roy AC, Cornthwaite KJ, LaPointe LC, Young GP. Detection of methylated BCAT1 and IKZF1 after curative-intent treatment as a prognostic indicator for colorectal cancer recurrence. Cancer Med. 2023; 12(2): 1319-1329.

[120] Khatami F, Larijani B, Nasiri S, Tavangar SM. Liquid biopsy as a minimally invasive source of thyroid cancer genetic and epigenetic alterations. Int J Mol Cell Med. 2019; 8: 19-29. Suppl 1.

[121] Dai L, Jing Z, Zhu Y, Deng K, Ma L. Genome-wide analysis of circulating tumor DNA methylation profiles in cerebrospinal fluid: a clinical trial of oncolytic virus for glioblastoma. Am J Cancer Res. 2023; 13(12): 5950-5965.

[122] Ehrlich M. DNA hypermethylation in disease: mechanisms and clinical relevance. Epigenetics. 2019; 14(12): 1141-1163.

[123] Klein EA, Richards D, Cohn A, et al. Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set. Ann Oncol. 2021; 32(9): 1167-1177.

[124] Bach S, Sluiter NR, Beagan JJ, et al. Circulating tumor DNA analysis: clinical implications for colorectal cancer patients. A systematic review. JNCI Cancer Spectr. 2019; 3(3): pkz042.

[125] Ko K, Kananazawa Y, Yamada T, et al. Methylation status and long-fragment cell-free DNA are prognostic biomarkers for gastric cancer. Cancer Med. 2021; 10(6): 2003-2012.

[126] Xu F, Yu S, Han J, et al. Detection of circulating tumor DNA methylation in diagnosis of colorectal cancer. Clin Transl Gastroenterol. 2021; 12(8): e00386.

[127] Brenne SS, Madsen PH, Pedersen IS, et al. Colorectal cancer detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study. Br J Cancer. 2023; 129(5): 861-868.

[128] Yu M, Moinova HR, Willbanks A, et al. Novel DNA methylation biomarker panel for detection of esophageal adenocarcinoma and high-grade dysplasia. Clin Cancer Res. 2022; 28(17): 3761-3769.

[129] Ooki A, Maleki Z, Tsay JJ, et al. A panel of novel detection and prognostic methylated DNA markers in primary non-small cell lung cancer and serum DNA. Clin Cancer Res. 2017; 23(22): 7141-7152.

[130] Liang W, Chen Z, Li C, et al. Accurate diagnosis of pulmonary nodules using a noninvasive DNA methylation test. J Clin Invest. 2021; 131(10): e145973.

[131] Bu Q, Luo X, He L, et al. Septin9 DNA methylation as a promising biomarker for cervical cancer. J Obstet Gynaecol. 2023; 43(1): 2151356.

[132] Gaga M, Chorostowska-Wynimko J, Horváth I, et al. Validation of Lung EpiCheck, a novel methylation-based blood assay, for the detection of lung cancer in European and Chinese high-risk individuals. Eur Respir J. 2021; 57(1): 2002682.

[133] Wever BMM, Bach S, Tibbesma M, et al. Detection of non-metastatic non-small-cell lung cancer in urine by methylation-specific PCR analysis: a feasibility study. Lung Cancer. 2022; 170: 156-164.

[134] Cai Z, Chen G, Zeng Y, et al. Comprehensive liquid profiling of circulating tumor DNA and protein biomarkers in long-term follow-up patients with hepatocellular carcinoma. Clin Cancer Res. 2019; 25(17): 5284-5294.

[135] Zhang H, Dong P, Guo S, et al. Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing. BMC Med. 2020; 18(1): 200.

[136] Chen MM, Zhao RC, Chen KF, et al. Hypomethylation of CTCFL promoters as a noninvasive biomarker in plasma from patients with hepatocellular carcinoma. Neoplasma. 2020; 67(4): 909-915.

[137] Hu N, Fan XP, Fan YC, et al. Hypomethylated ubiquitin-conjugating enzyme2 Q1 (UBE2Q1) gene promoter in the serum is a promising biomarker for hepatitis B virus-associated hepatocellular carcinoma. Tohoku J Exp Med. 2017; 242(2): 93-100.

[138] Zhang Q, Hu G, Yang Q, et al. A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA. Gynecol Oncol. 2013; 130(1): 132-139.

[139] Wang B, Yu L, Luo X, et al. Detection of OPCML methylation, a possible epigenetic marker, from free serum circulating DNA to improve the diagnosis of early-stage ovarian epithelial cancer. Oncol Lett. 2017; 14(1): 217-223.

[140] Dong R, Yu J, Pu H, Zhang Z, Xu X. Frequent SLIT2 promoter methylation in the serum of patients with ovarian cancer. J Int Med Res. 2012; 40(2): 681-686.

[141] Renard I, Joniau S, van Cleynenbreugel B, et al. Identification and validation of the methylated TWIST1 and NID2 genes through real-time methylation-specific polymerase chain reaction assays for the noninvasive detection of primary bladder cancer in urine samples. Eur Urol. 2010; 58(1): 96-104.

[142] Feber A, Dhami P, Dong L, et al. UroMark-a urinary biomarker assay for the detection of bladder cancer. Clin Epigenetics. 2017; 9: 8.

[143] Powrozek T, Krawczyk P, Kucharczyk T, Milanowski J. Septin 9 promoter region methylation in free circulating DNA-potential role in noninvasive diagnosis of lung cancer: preliminary report. Med Oncol. 2014; 31(4): 917.

[144] Church TR, Wandell M, Lofton-Day C, et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut. 2014; 63(2): 317-325.

[145] Luo H, Zhao Q, Wei W, et al. Circulating tumor DNA methylation profiles enable early diagnosis, prognosis prediction, and screening for colorectal cancer. Sci Transl Med. 2020; 12(524): eaax7533.

[146] Song L, Li Y, Jia J, et al. Algorithm optimization in methylation detection with multiple RT-qPCR. PLoS One. 2016; 11(11): e0163333.

[147] Xu RH, Wei W, Krawczyk M, et al. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat Mater. 2017; 16(11): 1155-1161.

[148] Jensen SO, Ogaard N, Nielsen HJ, Bramsen JB, Andersen CL. Enhanced performance of DNA methylation markers by simultaneous measurement of sense and antisense DNA strands after cytosine conversion. Clin Chem. 2020; 66(7): 925-933.

[149] Pedersen SK, Symonds EL, Baker RT, et al. Evaluation of an assay for methylated BCAT1 and IKZF1 in plasma for detection of colorectal neoplasia. BMC Cancer. 2015; 15: 654.

[150] Kisiel JB, Dukek BA, V S R Kanipakam R, et al. Hepatocellular carcinoma detection by plasma methylated DNA: discovery, phase I pilot, and phase II clinical validation. Hepatology. 2019; 69(3): 1180-1192.

[151] Ignatiadis M, Sledge GW, Jeffrey SS. Liquid biopsy enters the clinic—implementation issues and future challenges. Nat Rev Clin Oncol. 2021; 18(5): 297-312.

[152] Symonds EL, Pedersen SK, Murray D, et al. Circulating epigenetic biomarkers for detection of recurrent colorectal cancer. Cancer. 2020; 126(7): 1460-1469.

[153] Young GP, Pedersen SK, Mansfield S, et al. A cross-sectional study comparing a blood test for methylated BCAT1 and IKZF1 tumor-derived DNA with CEA for detection of recurrent colorectal cancer. Cancer Med. 2016; 5(10): 2763-2772.

[154] Pedersen SK, Musher BL, LaPointe LC, et al. Detection of recurrent colorectal cancer with high specificity using a reporting threshold for circulating tumor DNA methylated in BCAT1 and IKZF1. Cancer. 2022; 128(10): 1921-1928.

[155] Al Naji H, Winter JM, Pedersen SK, et al. Evaluating the role of methylated circulating tumor DNA in combination with pathological prognostic factors for predicting recurrence of colorectal cancer. Biomark Insights. 2024; 19: 11772719241232870.

[156] Sun J, Fei F, Zhang M, et al. The role of ^mSEPT9 in screening, diagnosis, and recurrence monitoring of colorectal cancer. BMC Cancer. 2019; 19(1): 450.

[157] Taieb J, Taly V, Vernerey D, et al. Analysis of circulating tumour DNA (ctDNA) from patients enrolled in the IDEA-FRANCE phase III trial: prognostic and predictive value for adjuvant treatment duration. Annals of Oncology. 2019; 30: P867.

[158] Slater S, Bryant A, Aresu M, et al. Tissue-free liquid biopsies combining genomic and methylation signals for minimal residual disease detection in patients with early colorectal cancer from the UK TRACC Part B study. Clin Cancer Res. 2024; 30(16): 3459-3469. Published online June 12, 2024.

[159] Lu CY, Chen SY, Peng HL, Kan PY, Chang WC, Yen CJ. Cell-free methylation markers with diagnostic and prognostic potential in hepatocellular carcinoma. Oncotarget. 2017; 8(4): 6406-6418.

[160] Su HY, Lai HC, Lin YW, Chou YC, Liu CY, Yu MH. An epigenetic marker panel for screening and prognostic prediction of ovarian cancer. Int J Cancer. 2009; 124(2): 387-393.

[161] Takahashi H, Kagara N, Tanei T, et al. Correlation of methylated circulating tumor DNA with response to neoadjuvant chemotherapy in breast cancer patients. Clin Breast Cancer. 2017; 17(1): 61-69. e63.

[162] Nakayama G, Kodera Y, Ohashi N, Koike M, Fujiwara M, Nakao A. p16INK4a methylation in serum as a follow-up marker for recurrence of colorectal cancer. Anticancer Res. 2011; 31(5): 1643-1646.

[163] Karamitrousis EI, Balgkouranidou I, Xenidis N, et al. Prognostic role of RASSF1A, SOX17 and Wif-1 promoter methylation status in cell-free DNA of advanced gastric cancer patients. Technol Cancer Res Treat. 2021; 20: 1533033820973279.

[164] Mastoraki S, Balgkouranidou I, Tsaroucha E, Klinakis A, Georgoulias V, Lianidou E. KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer. Mol Oncol. 2021; 15(9): 2412-2422.

[165] Henriksen SD, Madsen PH, Larsen AC, et al. Promoter hypermethylation in plasma-derived cell-free DNA as a prognostic marker for pancreatic adenocarcinoma staging. Int J Cancer. 2017; 141(12): 2489-2497.

[166] Khier S, Lohan L. Kinetics of circulating cell-free DNA for biomedical applications: critical appraisal of the literature. Future Sci OA. 2018; 4(4): FSO295.

[167] Boeckx N, Op de Beeck K, Beyens M, et al. Mutation and methylation analysis of circulating tumor DNA can be used for follow-up of metastatic colorectal cancer patients. Clin Colorectal Cancer. 2018; 17(2): e369-e379.

[168] Reece M, Saluja H, Hollington P, et al. The use of circulating tumor DNA to monitor and predict response to treatment in colorectal cancer. Front Genet. 2019; 10: 1118.

[169] Symonds EL, Pedersen SK, Yeo B, et al. Assessment of tumor burden and response to therapy in patients with colorectal cancer using a quantitative ctDNA test for methylated BCAT1/IKZF1. Mol Oncol. 2022; 16(10): 2031-2041.

[170] Bergheim J, Semaan A, Gevensleben H, et al. Potential of quantitative SEPT9 and SHOX2 methylation in plasmatic circulating cell-free DNA as auxiliary staging parameter in colorectal cancer: a prospective observational cohort study. Br J Cancer. 2018; 118(9): 1217-1228.

[171] Song L, Guo S, Wang J, et al. The blood mSEPT9 is capable of assessing the surgical therapeutic effect and the prognosis of colorectal cancer. Biomark Med. 2018; 12(9): 961-973.

[172] Chen H, Werner S, Tao S, Zornig I, Brenner H. Blood autoantibodies against tumor-associated antigens as biomarkers in early detection of colorectal cancer. Cancer Lett. 2014; 346(2): 178-187.

[173] Jiang D, Wang Y, Liu M, et al. A panel of autoantibodies against tumor-associated antigens in the early immunodiagnosis of lung cancer. Immunobiology. 2020; 225(1): 151848.

[174] Wen SWC, Andersen RF, Hansen TF, et al. The prognostic impact of circulating homeobox A9 methylated DNA in advanced non-small cell lung cancer. Transl Lung Cancer Res. 2021; 10(2): 855-865.

[175] Guo D, Yang L, Yang J, Shi K. Plasma cell-free DNA methylation combined with tumor mutation detection in prognostic prediction of patients with non-small cell lung cancer (NSCLC). Medicine (Baltimore). 2020; 99(26): e20431.

[176] Chimonidou M, Strati A, Malamos N, Georgoulias V, Lianidou ES. SOX17 promoter methylation in circulating tumor cells and matched cell-free DNA isolated from plasma of patients with breast cancer. Clin Chem. 2013; 59(1): 270-279.

[177] Chimonidou M, Tzitzira A, Strati A, et al. CST6 promoter methylation in circulating cell-free DNA of breast cancer patients. Clin Biochem. 2013; 46(3): 235-240.

[178] Balgkouranidou I, Chimonidou M, Milaki G, et al. Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer. Br J Cancer. 2014; 110(8): 2054-2062.

[179] Giannopoulou L, Mastoraki S, Buderath P, et al. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer. Gynecol Oncol. 2018; 150(2): 355-360.

[180] Zhou X, Jiao D, Dou M, et al. Association of glutathione-S-transferase p1 gene promoter methylation and the incidence of prostate cancer: a systematic review and meta-analysis. J Cancer Res Clin Oncol. 2019; 145(8): 1939-1948.

[181] Gurioli G, Martignano F, Salvi S, Costantini M, Gunelli R, Casadio V. GSTP1 methylation in cancer: a liquid biopsy biomarker?. Clin Chem Lab Med. 2018; 56(5): 702-717.

[182] Carson JJK, Di Lena MA, Berman DM, Siemens DR, Mueller CR. Development and initial clinical correlation of a DNA methylation-based blood test for prostate cancer. Prostate. 2020; 80(12): 1038-1042.

[183] Lak NSM, van Zogchel LMJ, Zappeij-Kannegieter L, et al. Cell-free DNA as a diagnostic and prognostic biomarker in pediatric rhabdomyosarcoma. JCO Precis Oncol. 2023; 7: e2200113.

[184] Fujita N, Nakayama T, Yamamoto N, et al. Methylated DNA and total DNA in serum detected by one-step methylation-specific PCR is predictive of poor prognosis for breast cancer patients. Oncology. 2012; 83(5): 273-282.

[185] Liggett TE, Melnikov AA, Marks JR, Levenson VV. Methylation patterns in cell-free plasma DNA reflect removal of the primary tumor and drug treatment of breast cancer patients. Int J Cancer. 2011; 128(2): 492-499.

[186] Herbst A, Vdovin N, Gacesa S, et al. Methylated free-circulating HPP1 DNA is an early response marker in patients with metastatic colorectal cancer. Int J Cancer. 2017; 140(9): 2134-2144.

[187] Huang ZH, Hu Y, Hua D, Wu YY, Song MX, Cheng ZH. Quantitative analysis of multiple methylated genes in plasma for the diagnosis and prognosis of hepatocellular carcinoma. Exp Mol Pathol. 2011; 91(3): 702-707.

[188] Rusan M, Andersen RF, Jakobsen A, Steffensen KD. Circulating HOXA9-methylated tumour DNA: a novel biomarker of response to poly (ADP-ribose) polymerase inhibition in BRCA-mutated epithelial ovarian cancer. Eur J Cancer. 2020; 125: 121-129.

[189] Symonds EL, Pedersen SK, Murray DH, et al. Circulating tumour DNA for monitoring colorectal cancer-a prospective cohort study to assess relationship to tissue methylation, cancer characteristics and surgical resection. Clin Epigenetics. 2018; 10: 63.

[190] Appelt AL, Andersen RF, Lindebjerg J, Jakobsen A. Prognostic value of serum NPY hypermethylation in neoadjuvant chemoradiotherapy for rectal cancer: secondary analysis of a randomized trial. Am J Clin Oncol. 2020; 43(1): 9-13.

[191] Melton CA, Freese P, Zhou Y, et al. A novel tissue-free method to estimate tumor-derived cell-free DNA quantity using tumor methylation patterns. Cancers (Basel). 2023; 16(1): 82.

[192] Angeli-Pahim I, Chambers A, Duarte S, et al. Methylated ctDNA quantification: noninvasive approach to monitoring hepatocellular carcinoma burden. J Am Coll Surg. 2024; 238(4): 770-778.

[193] Tran HT, Heeke S, Sujit S, et al. Circulating tumor DNA and radiological tumor volume identify patients at risk for relapse with resected, early-stage non-small-cell lung cancer. Ann Oncol. 2024; 35(2): 183-189.

[194] Riethdorf S, Soave A, Rink M. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer. Transl Androl Urol. 2017; 6(6): 1090-1110.

[195] Dor Y, Cedar H. Principles of DNA methylation and their implications for biology and medicine. Lancet. 2018; 392(10149): 777-786.

[196] Locke WJ, Guanzon D, Ma C, et al. DNA methylation cancer biomarkers: translation to the clinic. Front Genet. 2019; 10: 1150.

[197] Thompson MK, Gale D, Brenton JD. Circulating tumour DNA for clinicians: current and future clinical applications. Clin Radiol. 2021; 76(10): 737-747.

[198] Sun K, Jiang P, Chan KC, et al. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proc Natl Acad Sci USA. 2015; 112(40): E5503-5512.

[199] Gai W, Ji L, Lam WKJ, et al. Liver-and colon-specific DNA methylation markers in plasma for investigation of colorectal cancers with or without liver metastases. Clin Chem. 2018; 64(8): 1239-1249.

[200] Lyu X, Tsui YM, Ho DW, Ng IO. Liquid biopsy using cell-free or circulating tumor DNA in the management of hepatocellular carcinoma. Cell Mol Gastroenterol Hepatol. 2022; 13(6): 1611-1624.

[201] Wen SWC, Nederby L, Andersen RF, et al. NK cell activity and methylated HOXA9 ctDNA as prognostic biomarkers in patients with non-small cell lung cancer treated with PD-1/PD-L1 inhibitors. Br J Cancer. 2023; 129(1): 135-142.

[202] Clozel T, Yang S, Elstrom RL, et al. Mechanism-based epigenetic chemosensitization therapy of diffuse large B-cell lymphoma. Cancer Discov. 2013; 3(9): 1002-1019.

[203] Brem EA, Li H, Beaven AW, et al. SWOG 1918: a phase II/III randomized study of R-miniCHOP with or without oral azacitidine (CC-486) in participants age 75 years or older with newly diagnosed aggressive non-Hodgkin lymphomas—Aiming to improve therapy, outcomes, and validate a prospective frailty tool. J Geriatr Oncol. 2022; 13(2): 258-264.

[204] Luo R, Ge C, Xiao X, et al. Identification of genetic variations associated with drug resistance in non-small cell lung cancer patients undergoing systemic treatment. Brief Bioinform. 2021; 22(6): bbab187.

[205] Hicks JK, Bishop JR, Sangkuhl K, et al. Clinical pharmacogenetics implementation consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and dosing of selective serotonin reuptake inhibitors. Clin Pharmacol Ther. 2015; 98(2): 127-134.

[206] ctDNA Methylation for Epithelial Ovarian Cancer. https://www.clinicaltrials.gov/study/NCT05801263

[207] ctDNA Methylation for Detecting Ovarian Cancer. https://www.clinicaltrials.gov/study/NCT05801276

[208] ctDNA Methylation Sequencing for Myeloma. https://www.clinicaltrials.gov/study/NCT05578625

[209] ctDNA Methylation Application in Postoperative Relapse and Adjuvant Chemotherapy Efficacy Evaluation. https://www.clinicaltrials.gov/study/NCT05536089

[210] Comparison of Diagnostic Sensitivity Between ctDNA Methylation and CEA in Colorectal Cancer. https://www.clinicaltrials.gov/study/NCT05558436

[211] The Implication of Plasma ctDNA Methylation Haplotypes in Detecting Colorectal Cancer and Adenomas. https://www.clinicaltrials.gov/study/NCT03737591

[212] Clinical Validation of ColonAiQ (a Blood-based Assay Targeting ctDNA Methylation) for Colorectal Cancer Detection. https://www.clinicaltrials.gov/study/NCT06347887

[213] Diagnosis of Colorectal Cancer and Advanced Adenoma Using Cancer-specific Methylation Signatures. https://www.clinicaltrials.gov/study/NCT03828396

[214] Dynamic Monitoring of ctDNA Methylation to Predict Relapse in Colorectal Cancer After Radical Resection (POSTCA). https://www.clinicaltrials.gov/study/NCT03737539

[215] A Study on a Blood-based Dual-target Test for CRC Detection. https://www.clinicaltrials.gov/study/NCT05508503

[216] Circulating Tumor DNA Methylation Guided Postoperative Follow-up Strategy for Non-metastatic Colorectal Cancer. https://www.clinicaltrials.gov/study/NCT05904665

[217] Circulating Tumor DNA Methylation Guided Postoperative Adjuvant Chemotherapy for High-risk Stage II/III Colorectal Cancer. https://www.clinicaltrials.gov/study/NCT05954078

[218] Non-Invasive ctDNA Methylation Detection for Lung Nodule Patients. https://www.clinicaltrials.gov/study/NCT03685669

[219] Diagnostic and Monitoring Significance of ctDNA Methylation Analysis by NGS in Benign and Malignant Pulmonary Nodules. https://www.clinicaltrials.gov/study/NCT03651986

[220] Predicting Non-small Cell Lung Cancer (NSCLC) Lymph Node Metastasis: Integrating Circulating Tumor DNA (ctDNA) Mutation/Methylation Profiling With Positron Emission Tomography-computed Tomography (PET-CT) Scan. https://www.clinicaltrials.gov/study/NCT06358222

[221] Liquid Biopsy Using Methylation Sequencing for Lung Cancer. https://www.clinicaltrials.gov/study/NCT04253509

[222] Circulating Tumor DNA Methylation Test to Differentiate Benign and Malignant Pulmonary Nodules. https://www.clinicaltrials.gov/study/NCT03181490

[223] Monitoring of Circulating Tumor DNA and Its Aberrant Methylation in the Surveillance of Surgical Lung Cancer Patients (MEDAL, MEthylation Based Dynamic Analysis for Lung Cancer). (MEDAL). https://www.clinicaltrials.gov/study/NCT03634826

[224] HCC Screening Using DNA Methylation Changes in ctDNA. https://www.clinicaltrials.gov/study/NCT03483922

[225] Study on Early Auxiliary Diagnosis and Postoperative Recurrence Monitoring of Breast Cancer. https://www.clinicaltrials.gov/study/NCT05858242

[226] Exploration and Verification of DNA Methylolation in Early Screening of Esophageal Cancer. https://www.clinicaltrials.gov/study/NCT03922230

[227] Methylation Analysis of Circulating Tumor DNA in Gastric Cancer. https://www.clinicaltrials.gov/study/NCT04511559

[228] Use of Plasma ctDNA Methylation Haplotypes in Detecting Local Residual or Lymph Node Metastasis. https://www.clinicaltrials.gov/study/NCT03868215

[229] Circulating Tumor DNA Methylation Guiding Postoperative Adjuvant Chemotherapy in Stage III Colorectal Cancer (cmPAT). https://www.clinicaltrials.gov/study/NCT06167967

[230] Andersen RF. Tumor-specific methylations in circulating cell-free DNA as clinically applicable markers with potential to substitute mutational analyses. Expert Rev Mol Diagn. 2018; 18(12): 1011-1019.

[231] Shen SY, Singhania R, Fehringer G, et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature. 2018; 563(7732): 579-583.

[232] Cree IA, Uttley L, Buckley Woods H, et al. The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review. BMC Cancer. 2017; 17(1): 697.

[233] Ossandon MR, Agrawal L, Bernhard EJ, et al. Circulating tumor DNA assays in clinical cancer research. J Natl Cancer Inst. 2018; 110(9): 929-934.

[234] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019; 69(1): 7-34.

[235] Kang G, Chen K, Yang F, et al. Monitoring of circulating tumor DNA and its aberrant methylation in the surveillance of surgical lung Cancer patients: protocol for a prospective observational study. BMC Cancer. 2019; 19(1): 579.

[236] Tao Y, Xing S, Zuo S, et al. Cell-free multi-omics analysis reveals potential biomarkers in gastrointestinal cancer patients’ blood. Cell Rep Med. 2023; 4(11): 101281.

[237] Boriachek K, Islam MN, Möller A, et al. Biological functions and current advances in isolation and detection strategies for exosome nanovesicles. Small. 2018; 14(6): 1702153.

[238] Singh K, Nalabotala R, Koo KM, Bose S, Nayak R, Shiddiky MJA. Separation of distinct exosome subpopulations: isolation and characterization approaches and their associated challenges. Analyst. 2021; 146(12): 3731-3749.

[239] Mouliere F, Chandrananda D, Piskorz AM, et al. Enhanced detection of circulating tumor DNA by fragment size analysis. Sci Transl Med. 2018; 10(466): eaat4921.

[240] Zhang C, Leng W, Sun C, et al. Urine proteome profiling predicts lung cancer from control cases and other tumors. EBioMedicine. 2018; 30: 120-128.

[241] van Huizen NA, van Rosmalen J, Dekker LJM, et al. Identification of a collagen marker in urine improves the detection of colorectal liver metastases. J Proteome Res. 2020; 19(1): 153-160.

[242] Mezzalira S, De Mattia E, Guardascione M, Dalle Fratte C, Cecchin E, Toffoli G. Circulating-free DNA analysis in hepatocellular carcinoma: a promising strategy to improve patients’ management and therapy outcomes. Int J Mol Sci. 2019; 20(21): 5498.

[243] Warren JD, Xiong W, Bunker AM, et al. Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer. BMC Med. 2011; 9: 133.

[244] Zhao Z, Cai S, Wang Z. Circulating tumor DNA as a prognostic marker in stage III colon cancer. JAMA Oncol. 2020; 6(6): 932.

[245] Chen X, Zhang J, Ruan W, et al. Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer. J Clin Invest. 2020; 130(12): 6278-6289.

[246] Pantel K, Alix-Panabieres C. Liquid biopsy and minimal residual disease—latest advances and implications for cure. Nat Rev Clin Oncol. 2019; 16(7): 409-424.

[247] Lu P, Zhu X, Song Y, et al. Methylated septin 9 as a promising biomarker in the diagnosis and recurrence monitoring of colorectal cancer. Dis Markers. 2022; 2022: 7087885.

[248] Potter NT, Hurban P, White MN, et al. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma. Clin Chem. 2014; 60(9): 1183-1191.

[249] Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early-and late-stage human malignancies. Sci Transl Med. 2014; 6(224): 224ra224.

[250] Wang Y, Springer S, Zhang M, et al. Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci USA. 2015; 112(31): 9704-9709.

RIGHTS & PERMISSIONS

2024 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.