Unleashing the potential of urine DNA methylation detection: Advancements in biomarkers, clinical applications, and emerging technologies

Abstract

In recent years, the detection urinary DNA methylation in bladder cancer has witnessed significant advancements. Important breakthroughs have been achieved in the diagnosis of bladder cancer through the use of DNA methylation biomarkers in urine. Several clinical studies have successfully established multiple biomarkers and developed reliable diagnostic models. Additionally, certain assay kits are certified by the Food and Drug Administration or the National Medical Products Administration and provide dependable tools for clinical applications. However, traditional techniques have limitations in terms of sample requirements, operational complexity, and stability. This review presents the application of novel technologies for the detection of urinary DNA methylation in bladder cancer, including microfluidic, digital polymerase chain reaction, and CRISPR technologies. The introduction of these innovative approaches holds promise for enhancing the early diagnosis and prognosis of bladder cancer. These advances are expected to drive further research and clinical applications in this field.

Keywords

DNA methylation / Urine / Microfluidics / Bladder cancer / Digital PCR

Acknowledgments

None.

Statement of ethics

None.

Conflict of interest statement

DT is an editor-in-chief, JL is an associate editor, FW is an editorial board member of Current Urology. They confirm no involvement in any stage of this article’s peer-review process, ensuring unbiased editorial decision-making. This article was accepted after normal external review. No conflict of interest has been declared by the other authors.

Funding source

None

Author contributions

XS: Writing of the manuscript;

SZ, JW, DW: Consultation of the information;

HN, FW: Embellishment of the language;

DT, JL: Review of the manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.

References

[1] Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin 2022; 72(1):7-33.

[2] Bouchelouche K. PET/CT in bladder cancer: An update. Semin Nucl Med 2022; 52(4):475-485.

[3] Taylor J, Becher E, Steinberg GD. Update on the guideline of guidelines: Non-muscle-invasive bladder cancer. BJU Int 2020; 125(2):197-205.

[4] Kartolo A, Kassouf W, Vera-Badillo FE. Adjuvant immune checkpoint inhibition in muscle-invasive bladder cancer: Is it ready for prime time? Eur Urol 2021; 80(6):679-681.

[5] Matuszczak M, Kiljańczyk A, Salagierski M. A liquid biopsy in bladder cancer-The current landscape in urinary biomarkers. Int J Mol Sci 2022; 23(15):8597.

[6] van der Aa MN, Steyerberg EW, Sen EF, et al. Patients' perceived burden of cystoscopic and urinary surveillance of bladder cancer: A randomized comparison. BJU Int 2008; 101(9):1106-1110.

[7] van Rhijn BW, van der Poel HG, van der Kwast TH. Urine markers for bladder cancer surveillance: A systematic review. Eur Urol 2005; 47(6):736-748.

[8] Teixeira-Marques A, Lourenço C, Oliveira MC, Henrique R, Jerónimo C. Extracellular vesicles as potential bladder cancer biomarkers: Take it or leave it? Int J Mol Sci 2023; 24(7):6757.

[9] Im DH, Peng CC, Xu L, et al. Potential of aqueous humor as a liquid biopsy for uveal melanoma. Int J Mol Sci 2022; 23(11):6226.

[10] Vollbrecht C, Hoffmann I, Lehmann A, et al. Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue. Virchows Arch 2023; 482(4):697-706.

[11] Rapado-González Ó, Martínez-Reglero C, Salgado-Barreira Á, et al. Salivary biomarkers for cancer diagnosis: A meta-analysis. Ann Med 2020; 52(3-4):131-144.

[12] Pagès M, Rotem D, Gydush G, et al. Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine. Neuro Oncol 2022; 24(8):1352-1363.

[13] Sabato C, Noviello TMR, Covre A, et al. A novel microRNA signature for the detection of melanoma by liquid biopsy. J Transl Med 2022; 20(1):469.

[14] Deng L, Chao H, Deng H, et al. A novel and sensitive DNA methylation marker for the urine-based liquid biopsies to detect bladder cancer. BMC Cancer 2022; 22(1):510.

[15] Bergantim R, Peixoto da Silva S, Polónia B, et al. Detection of measurable residual disease biomarkers in extracellular vesicles from liquid biopsies of multiple myeloma patients-A proof of concept. Int J Mol Sci 2022; 23(22):13686.

[16] Yunusova N, Dzhugashvili E, Yalovaya A, et al. Comparative analysis of tumor-associated microRNAs and tetraspanines from exosomes of plasma and ascitic fluids of ovarian cancer patients. Int J Mol Sci 2022; 24(1):464.

[17] Krol I, Schwab FD, Carbone R, et al. Detection of clustered circulating tumour cells in early breast cancer. Br J Cancer 2021; 125(1):23-27.

[18] El-Falouji AI, Sabri DM, Lotfi NM, et al. Rapid detection of recurrent non-muscle invasive bladder cancer in urine using ATR-FTIR technology. Molecules 2022; 27(24):8890.

[19] Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005; 6(8):597-610.

[20] Yong WS, Hsu FM, Chen PY. Profiling genome-wide DNA methylation. Epigenetics Chromatin 2016;9:26.

[21] Zhang X, Yazaki J, Sundaresan A, et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 2006; 126(6):1189-1201.

[22] Chen C, Wang Z, Ding Y, et al. DNA methylation: From cancer biology to clinical perspectives. Front Biosci (Landmark Ed) 2022; 27(12):326.

[23] Jones PA. Functions of DNA methylation: Islands, start sites, gene bodies and beyond. Nat Rev Genet 2012; 13(7):484-492.

[24] Esteller M. Epigenetics in cancer. N Engl J Med 2008; 358(11):1148-1159.

[25] Andersson R, Gebhard C, Miguel-Escalada I, et al. An atlas of active enhancers across human cell types and tissues. Nature 2014; 507(7493):455-461.

[26] Nunes SP, Henrique R, Jerónimo C, Paramio JM. DNA methylation as a therapeutic target for bladder cancer. Cells 2020; 9(8):1850.

[27] Larsen LK, Lind GE, Guldberg P, Dahl C. DNA-methylation-based detection of urological cancer in urine: Overview of biomarkers and considerations on biomarker design, source of DNA, and detection technologies. Int J Mol Sci 2019; 20(11):2657.

[28] Kandimalla R, van Tilborg AA, Zwarthoff EC. DNA methylation-based biomarkers in bladder cancer. Nat Rev Urol 2013; 10(6):327-335.

[29] Beukers W, Kandimalla R, Masius RG, et al. Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer. Mod Pathol 2015; 28(4):515-522.

[30] Wu Z, Xia C, Zhang C, Yang D, Ma K. Prognostic significance of SNCA and its methylation in bladder cancer. BMC Cancer 2022; 22(1):330.

[31] Chen JQ, Salas LA, Wiencke JK, et al. Immune profiles and DNA methylation alterations related with non-muscle-invasive bladder cancer outcomes. Clin Epigenetics 2022; 14(1):14.

[32] Carrot-Zhang J, Chambwe N, Damrauer JS, et al. Comprehensive analysis of genetic ancestry and its molecular correlates in cancer. Cancer Cell 2020; 37(5):639-654.e6.

[33] Bunch B, Krishnan N, Greenspan RD, et al. TAp73 expression and P1 promoter methylation, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle-invasive bladder cancer (MIBC). Cell Cycle 2019; 18(17):2055-2066.

[34] Piatti P, Chew YC, Suwoto M, et al. Clinical evaluation of bladder CARE, a new epigenetic test for bladder cancer detection in urine samples. Clin Epigenetics 2021; 13(1):84.

[35] Rose M, Bringezu S, Godfrey L, et al. ITIH5 and ECRG4 DNA methylation biomarker test (EI-BLA) for urine-based non-invasive detection of bladder cancer. Int J Mol Sci 2020; 21(3):1117.

[36] 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.

[37] Ruan W, Chen X, Huang M, et al. A urine-based DNA methylation assay to facilitate early detection and risk stratification of bladder cancer. Clin Epigenetics 2021; 13(1):91.

[38] Hentschel AE, Beijert IJ, Bosschieter J, et al. Bladder cancer detection in urine using DNA methylation markers: A technical and prospective preclinical validation. Clin Epigenetics 2022; 14(1):19.

[39] Wu Y, Jiang G, Zhang N, et al. HOXA9, PCDH17, POU4F2, and ONECUT2 as a urinary biomarker combination for the detection of bladder cancer in Chinese patients with hematuria. Eur Urol Focus 2020; 6(2):284-291.

[40] van Kessel KE, Beukers W, Lurkin I, et al. Validation of a DNA methylation-mutation urine assay to select patients with hematuria for cystoscopy. J Urol 2017; 197(3 Pt1):590-595.

[41] Feng Y, Jiang Y, Feng Q, et al. A novel prognostic biomarker for muscle invasive bladder urothelial carcinoma based on 11 DNA methylation signature. Cancer Biol Ther 2020; 21(12):1119-1127.

[42] Keeley B, Stark A, Pisanic TR 2nd, et al. Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events. Clin Chim Acta 2013;425:169-175.

[43] Yoon J, Park MK, Lee TY, Yoon YJ, Shin Y. LoMA-B: A simple and versatile lab-on-a-chip system based on single-channel bisulfite conversion for DNA methylation analysis. Lab Chip 2015; 15(17):3530-3539.

[44] Lee TY, Shin Y, Park MK. A simple, low-cost, and rapid device for a DNA methylation-specific amplification/detection system using a flexible plastic and silicon complex. Lab Chip 2014; 14(21):4220-4229.

[45] Ronen M, Avrahami D, Gerber D. A sensitive microfluidic platform for a high throughput DNA methylation assay. Lab Chip 2014; 14(13):2354-2362.

[46] Pharo HD, Jeanmougin M, Ager-Wick E, et al. BladMetrix: A novel urine DNA methylation test with high accuracy for detection of bladder cancer in hematuria patients. Clin Epigenetics 2022; 14(1):115.

[47] Hermanns T, Savio AJ, Olkhov-Mitsel E, et al. A noninvasive urine-based methylation biomarker panel to detect bladder cancer and discriminate cancer grade. Urol Oncol 2020; 38(6):603.e1-603.e7.

[48] Hentschel AE, Nieuwenhuijzen JA, Bosschieter J, et al. Comparative analysis of urine fractions for optimal bladder cancer detection using DNA methylation markers. Cancers (Basel) 2020; 12(4):859.

[49] Hentschel AE, van den Helder R, van Trommel NE, et al. The origin of tumor DNA in urine of urogenital cancer patients: Local shedding and transrenal excretion. Cancers (Basel) 2021; 13(3):535.

[50] Mijnes J, Veeck J, Gaisa NT, et al. Promoter methylation of DNA damage repair (DDR) genes in human tumor entities: RBBP8/CtIP is almost exclusively methylated in bladder cancer. Clin Epigenetics 2018;10:15.

[51] Roperch JP, Hennion C. A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients—Design of the Urodiag® PCR kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC). BMC Med Genet 2020; 21(1):112.

[52] Bosschieter J, et al. A two-gene methylation signature for the diagnosis of bladder cancer in urine. Epigenomics vol 2019; 11(3):337-347.

[53] van der Heijden AG, Mengual L, Ingelmo-Torres M, et al. Urine cell-based DNA methylation classifier for monitoring bladder cancer. Clin Epigenetics 2018;10:71.

[54] Georgopoulos P, et al. DNA hypermethylation of a panel of genes as an urinary biomarker for bladder cancer diagnosis. Urol J 2021; 19(3):214-220.

[55] Oh TJ, et al. Identification and validation of methylated PENK gene for early detection of bladder cancer using urine DNA. BMC Cancer 2022; 22(1):1195.

[56] Zhang J, et al. A novel methylation marker NRN1 plus TERT and FGFR3 mutation using urine sediment enables the detection of urothelial bladder carcinoma. Cancers 2023; 15(3):615.

[57] 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.

[58] Mancini M, Righetto M, Zumerle S, Montopoli M, Zattoni F. The bladder epiCheck test as a non-invasive tool based on the identification of DNA methylation in bladder cancer cells in the urine: A review of published evidence. Int J Mol Sci 2020; 21(18):6542.

[59] Trenti E, D'Elia C, Mian C, et al. Diagnostic predictive value of the bladder EpiCheck test in the follow-up of patients with non-muscle-invasive bladder cancer. Cancer Cytopathol 2019; 127(7):465-469.

[60] Pierconti F, Martini M, Cenci T, et al. The bladder epicheck test and cytology in the follow-up of patients with non-muscle-invasive high grade bladder carcinoma. Urol Oncol 2022; 40(3):108.e19-108.e25.

[61] Pierconti F, Martini M, Fiorentino V, et al. Upper urothelial tract high-grade carcinoma: Comparison of urine cytology and DNA methylation analysis in urinary samples. Hum Pathol 2021;118:42-48.

[62] Pierconti F, Martini M, Cenci T, et al. Methylation study of the Paris system for reporting urinary (TPS) categories. J Clin Pathol 2021; 74(2):102-105.

[63] D'Andrea D, Soria F, Zehetmayer S, et al. Diagnostic accuracy, clinical utility and influence on decision-making of a methylation urine biomarker test in the surveillance of non-muscle-invasive bladder cancer. BJU Int 2019; 123(6):959-967.

[64] Reinert T, Borre M, Christiansen A, Hermann GG, Ørntoft TF, Dyrskjøt L. Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation. PloS One 2012; 7(10):e46297.

[65] Zhang N, Chen S, Wu L, et al. Identification of cancer-specific methylation of gene combination for the diagnosis of bladder cancer. J Cancer 2019; 10(26):6761-6766.

[66] Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—Occurrence, properties and removal. J Appl Microbiol 2012; 113(5):1014-1026.

[67] O'Keefe CM, Giammanco D, Li S, Pisanic TR, Wang TJ. Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation. Lab Chip 2019; 19(3):444-451.

[68] Han K, Lee TY, Nikitopoulos DE, Soper SA, Murphy MC. A vertically stacked, polymer, microfluidic point mutation analyzer: Rapid high accuracy detection of low-abundance K-ras mutations. Anal Biochem 2011; 417(2):211-219.

[69] Kojabad AA, Farzanehpour M, Galeh HEG, et al. Droplet digital PCR of viral DNA/RNA, current progress, challenges, and future perspectives. J Med Virol 2021; 93(7):4182-4197.

[70] Pinheiro LB, Coleman VA, Hindson CM, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 2012; 84(2):1003-1011.

[71] Wiencke JK, Bracci PM, Hsuang G, et al. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood. Epigenetics 2014; 9(10):1360-1365.

[72] Pharo HD, Andresen K, Berg KCG, Lothe RA, Jeanmougin M, Lind GE. A robust internal control for high-precision DNA methylation analyses by droplet digital PCR. Clin Epigenetics 2018;10:24.

[73] Yager P, Edwards T, Fu E, et al. Microfluidic diagnostic technologies for global public health. Nature 2006; 442(7101):412-418.

[74] 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.

[75] Beinse G, Borghese B, Métairie M, et al. Highly specific droplet-digital PCR detection of universally methylated circulating tumor DNA in endometrial carcinoma. Clin Chem 2022; 68(6):782-793.

[76] Manco L, Dias HC. DNA methylation analysis of ELOVL2 gene using droplet digital PCR for age estimation purposes. Forensic Sci Int 2022;333:111206.

[77] Li N, Dhilipkannah P, Jiang F. High-throughput detection of multiple miRNAs and methylated DNA by droplet digital PCR. J Pers Med 2021; 11(5):359.

[78] Peter MR, Bilenky M, Shi Y, et al. A novel methylated cell-free DNA marker panel to monitor treatment response in metastatic prostate cancer. Epigenomics 2022; 14(13):811-822.

[79] Wang JY, Doudna JA. CRISPR technology: A decade of genome editing is only the beginning. Science 2023; 379(6629):eadd8643.

[80] Li L, Li S, Wu N, et al. HOLMESv2: A CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. ACS Synth Biol 2019; 8(10):2228-2237.