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.

1. Introduction

Bladder cancer (BCa) is a widespread global malignancy, ranking as the fourth most prevalent cancer among men. Furthermore, it is the second most prevalent urological tumor following prostate cancer.^[1] Non–muscle-invasive BCa (NMIBC) accounts for approximately 75% of BCa cases and is the most prevalent form of BCa. It manifests as a superficial tumor with a high propensity for recurrence. However, it is generally not considered life threatening.^[2,3] Non–muscle-invasive BCa has been documented to exhibit a 5-year recurrence rate of up to 70% and a 5-year progression rate of up to 30%.^[3] Muscle-invasive BCa (MIBC) shows a significant tendency to spread to the lymph nodes and other organs. Approximately half of patients with MIBC develop metastases, leading to a poor prognosis, with many dying of the disease within 3 years.^[4] Once NMIBC progresses to muscle invasion and develops to MIBC, it can significantly affect patient prognosis.^[5] Therefore, regular, and effective monitoring of patients with BCa is crucial for the early detection of any signs of progression. Timely intervention and appropriate treatment can improve the long-term survival of patients diagnosed with BCa. Early detection and careful surveillance play vital roles in disease management and enhancing patient outcomes.

Invasive cystoscopy is the criterion standard for diagnosing and monitoring BCa. However, the high cost, discomfort, and infection risk associated with this procedure limit its widespread applicability.^[6] Urine cytology is a noninvasive alternative to cystoscopy with high specificity but limited sensitivity of only 17%, particularly for low-grade (LG) tumors.^[5,7] Thus, there is a critical need for a highly specific and sensitive noninvasive liquid biopsy marker for BCa diagnosis. Efforts are underway to develop such markers to improve accuracy and enable early detection, ultimately enhancing patient outcomes.

In recent years, liquid biopsies have seen rapid development as noninvasive biomarker test for early diagnosis and real-time disease surveillance. This advancement represents a significant innovation in the field of precision medicine.^[8] Liquid biopsies offer a noninvasive or minimally invasive sample collection from various body fluids, such as blood, urine, saliva, cerebrospinal fluid, and pleural fluid. This approach allows convenient and accessible sampling and provides valuable molecular information for diagnostic and monitoring purposes.^[9–12] Extensive research supports the use of circulating tumor cells, DNA, RNA, proteins, and exosomes in body fluids as cancer biomarkers. These biomarkers offer valuable insights into the diagnosis, prognosis, and monitoring of cancer. They enable personalized and targeted approaches for cancer treatment.^[13–17] With its noninvasive sampling, high reproducibility, excellent tumor specificity, and effective monitoring, urine liquid biopsy has the potential to replace cystoscopy in bladder tumor follow-up.^[18] In recent years, advancements in urine liquid biopsy have led to the utilization of various tumor markers obtained from urine samples for the diagnosis and detection of BCa. Notably, analysis of methylated DNA in urine has emerged as a promising diagnostic tool for BCa. It offers high specificity and sensitivity while being cost-effective and easily applicable, rendering it a prominent area of research in the field.

Methylation, an epigenetic modification, plays a crucial role in regulating gene expression without changing the underlying DNA sequence; thus, methylation-based techniques are of considerable interest in all fields of pathology.^[19] DNA methylation involves the addition of a methyl group from S-adenosylmethionine to the fifth carbon of cytosine (C) to form 5-methylcytosine (5mC), catalyzed by DNA methyltransferase.^[20] Methylation occurs predominantly in CpG islands, which are primarily located in the promoter region of the genome. CpG islands are characterized by a high density of CpG dinucleotides.^[21] DNA methylation is involved in several physiological and cellular processes. For example, it plays a critical role in transcriptional silencing by preventing the expression of specific genes in certain tissues.^[22] Additionally, DNA methylation is essential for regulating embryonic development, genomic imprinting, chromatin structural changes, and transposon inactivation.^[22,23] Abnormal DNA methylation levels are closely linked to tumor development and are detectable in the early stages of tumor formation. Typically, tumors exhibit genome-wide DNA hypomethylation, along with focal DNA hypermethylation at CpG-enriched sites. Research has demonstrated that DNA hypermethylation in the promoter region of the genome leads to the silencing of tumor suppressor genes, contributing to tumorigenesis.^[24] In addition to the substantial variations in methylation levels observed between tumor and normal tissues, notable differences in methylation patterns exist among different tumor types.^[25,26] Therefore, alterations in methylation levels hold great promise as specific molecular biomarkers for the detection and prognosis of BCa.^[26–28]

In recent years, researchers have extensively studied urinary DNA methylation as a noninvasive biomarker for BCa. The scientific validity of methylation markers as diagnostic and prognostic biomarkers in BCa at the genetic level has been highlighted in several studies, laying the foundation for the clinical application of this technology.^[29–33] Researchers have identified a large number of methylation sites through sequencing and bioinformatics analysis. They used techniques such as bisulfite sequencing, methylation-specific polymerase chain reaction (MSP), or methylation-specific restriction endonucleases^[34] to detect and analyze the methylation levels of these markers in the urine of patients with BCa. Results revealed the identification of several DNA methylation sites with high sensitivity and specificity for BCa. These loci have been utilized to construct multiple diagnostic and prognostic models of BCa.^[14,35–41] However, the high cost and complexity of the current detection process limit its potential for widespread clinical applications. To address this issue, researchers have explored the integration of conventional detection methods with emerging technologies, such as microfluidic chips as well as thermostatic and digital polymerase chain reactions (PCRs). The aim was to develop a more accurate, rapid, and user-friendly approach for assessing urinary DNA methylation levels in patients.^[42–46]

To summarize the advancements in the field of urinary DNA methylation detection for BCa, we conducted a comprehensive review of relevant literature from the past 5 years (2018 to present). We analyzed a significant number of studies that focused on the selection of methylation sites and their diagnostic performance. We also emphasized the innovative combinations of traditional MSP and emerging technologies. Moreover, we briefly introduced a few urinary DNA methylation biomarkers for BCa that have been applied in routine clinical practice.

2. Conventional technical process

Through extensive research and clinical practice, the urinary DNA methylation detection technology has evolved into a relatively mature workflow. This workflow included the collection of urine samples from the patients, extraction and processing of DNA, detection of DNA methylation levels, and subsequent data analyses (Fig. 1).

Figure 1.

The process of urine DNA methylation detection techniques. (A) Urine sample collection. (B) Centrifugation of urine samples.(C) DNA extraction. (D) Bisulfite processing in DNA. (E) Methylation-specific quantitative real-time PCR. PCR = polymerase chain reaction.

2.1. Urine sample collection

Researchers generally collect urine samples from patients suspected of having BCa (based on symptoms such as hematuria or abnormal urinary tract imaging) at hospitals. According to the diagnostic results, the patients were divided into 2 groups: the positive BCa group (BCA), which was diagnosed through cystoscopy or intraoperative pathological examination, and the non-BCA group, which includes patients who have been diagnosed with conditions such as urinary system stones, urinary tract infections, or benign urinary system disorders.^[34,37] Notably, the patient’s urine should be collected prior to cystoscopy or transurethral resection of the bladder tumor (TURBT). Typically, approximately 50 mL of urine is collected in the morning to ensure sufficient amount of high-quality DNA. After collection, the urine is usually processed within 24 to 72 hours by centrifugation to obtain the urine sediment. The sediment is subsequently resuspended using phosphate-buffered saline and stored at −20°C or −80°C for further analysis.^{[37–41,47,48]} Hentschel et al.^[48] conducted a study to evaluate which urine component was most suitable for the diagnosis of BCa using DNA methylation markers. Previous research has mainly focused on urine particles, whereas their study assessed the suitability of entire urine, urine particles, and supernatant. They measured the methylation levels of 9 markers in these 3 urine components and found that all 3 urine fractions were suitable for urine methylation analysis. For most markers, the methylation levels in each urine component were significantly correlated with the corresponding tumor tissue.^[48] Urine particles were the most representative fraction. Importantly, they demonstrated for the first time that urine supernatant is also applicable.^[48,49] In another study, Hentschel et al.^[49] collected urine samples from patients with BCa during natural voiding and from urine samples obtained to avoid direct contact with local tumors (e.g., postnephrostomy urine). The supernatant was collected by centrifugation, and DNA was extracted from the supernatants of both urine types. Methylation levels of specific biomarkers were analyzed. Through comparative analysis, they found that methylation levels were detected not only in urine samples obtained during natural voiding of patients with BCa, but also in the corresponding postnephrostomy urine and urine obtained from a catheter. This suggests that urinary DNA originates from the local shedding of tumor cells and cell fragments, as well as tumor DNA excreted through the kidneys.^[49] In addition, certain laboratories or companies such as the Pangea Laboratory^[34] utilize the Bladder CARE urine collection kit to obtain urine samples from healthy controls through mail-in services. Stable urine samples can be preserved at room temperature for up to 1 month.

2.2. DNA extraction, bisulfite treatment, and methylation analysis

The objective of DNA extraction is to effectively isolate and purify nucleic acids from urine samples. This can be achieved by utilizing commercial DNA extraction kits such as the QIAamp DNA Blood Mini Kit (catalog no. 51106; Qiagen, Hilden, Germany) or the Quick-DNA Urine Kit (D3061; Zymo Research, California, USA), among others.^[36] Alternative methods can be used for this purpose. Once the DNA is successfully separated, it is essential to quantify the extracted DNA and standardize its quantity to a consistent amount for each sample, ensuring integrity of the subsequent analyses is not compromised.

Methylation-specific PCR detection requires the conversion of extracted DNA through sodium bisulfite treatment. This crucial step involves the chemical modification of unmethylated cytosines in the DNA, converting them into sulfurated cytosines while leaving methylated cytosines unaffected. The purpose of this conversion process is to selectively amplify the methylated regions of DNA during PCR. Notably, the bisulfite treatment is not always required. Piatti et al.^[34] developed the Bladder CARE technique, which utilizes the Methylation-Sensitive Restriction Enzyme assay. This method allows for direct purification and analysis of DNA extracted from urine samples, eliminating the need for bisulfite treatment. The Methylation-Sensitive Restriction Enzyme assay specifically targets methylated DNA regions, enabling a streamlined approach for DNA purification and direct analysis from urine samples.

After the completion of bisulfite conversion, some researchers have used the MSP method to quantitatively measure DNA methylation levels of target markers. Mijnes et al.^[50] utilized MSP and bisulfite pyrosequencing to target the region near the transcription start site to assess the methylation status of retinoblastoma binding protein 8 in the urine and cell line DNA of patients with BCa. Experimental findings have revealed that retinoblastoma binding protein 8 could serve as a highly specific biomarker, indicating its potential utility in diagnostic applications. However, the conventional MSP lacks quantitative capabilities. Therefore, most researchers opted for multiplex fluorescent real-time quantitative PCR (qPCR) systems to precisely measure the methylation levels of target markers. This approach enables the accurate quantification of DNA methylation and offers valuable insights for the detection of BCa.^[37,38,51]

3. Performance of DNA methylation biomarkers

The selection of appropriate gene loci for analysis is of paramount importance in urinary DNA methylation testing for BCa. These loci could serve as effective biomarkers for the diagnosis and monitoring of BCa. Researchers typically use public databases, such as TCGA and GEO, to screen for potential BCa-specific DNA methylation sites. Prior to subsequent experimental validation, some investigators also performed targeted methylation sequencing of CpG loci. Chen et al.^[36] utilized the cutting-edge AnchorIRIS technology to perform targeted methylation sequencing of 100,000 CpG loci. Additionally, the single nucleotide polymorphism genotyping MassARRAY system was used to assess the methylation status of multiple CpG loci in diverse genomic regions. Through a comprehensive analysis, they identified 26 robustly methylated markers for BCa and established a diagnostic model named utMeMA, centered on the 2 markers OTX1/SOX1-OT. This model demonstrated exceptional accuracy (86.7%), sensitivity (90.0%), and specificity (83.1%) in BCa patients, underscoring its clinical relevance and potential utility.^[36] Table 1 summarizes individual DNA methylation biomarkers and marker combinations with high sensitivity in BCA and provides data on their sensitivity and specificity. Notably, the diagnostic performance of the same gene locus may vary depending on the detection method used; hence, the data should be interpreted with caution. However, we believe that, with the application of emerging technologies for specific methylation detection, the efficiency of diagnostic models constructed using these biomarkers will be further improved.

Table 1.

DNA methylation biomarkers with promising performance in recent research.

Authors	Gene symbol	n	Year of publication	Sensitivity, %	Specificity, %	PPV, %	NPV, %
Chen et al.[36]	OTX1/SOX1-OT	488	2020	90	83.1	‐	‐
Chen et al.[36]	ONECUT2/VIM	464	2021	88.1–91.2	85.7–89.7	‐	‐
Hentschel et al.[38]	GHSR/MAL	208	2022	80	93	‐	‐
Bosschieter et al.[52]	GHSR/MAL	147	2019	92	85	‐	‐
Piatti et al.[34]	TRNA-Cys/SIM2/NKX1–1	213	2021	93.5	92.6	‐	‐
Hermanns et al.[47]	NID2/Twist1	313	2020	‐	‐	81	55
Wu et al.[39]	HOXA9/PCDH17/POU4F2/ONECUT2	192	2020	‐	‐	100	84
Van der Heijde et al.[53]	CFTR/SALL3/TWIST1	626	2018	96	40	56	92
Georgopoulos et al.[54]	RASSF1/DAPK/RARB/TERT/APC/ACTB	85	2021	61.4	86.4	90	53
Oh et al.[55]	PENK	224	2022	86.5 (High-grade tumor)	92.5 (High-grade tumor)	‐	‐
Zhang et al.[56]	NRN1/TERT C228T/FGFR3 p.S249C	443	2023	81.7	89.5	‐	‐

NPV = negative predictive value; PPV = positive predictive value.

4. Partial urinary DNA methylation-based bladder cancer test kits approved by the Food and Drug Administration and the National Medical Products Administration

Several urinary DNA methylation-based BCa test kits have been approved by the US Food and Drug Administration (FDA) and the National Medical Products Administration (NMPA) of China. These test kits have demonstrated significant detection efficacy and accuracy after rigorous clinical research validation. The approved DNA methylation biomarkers provide reliable tools for the early screening and diagnosis of BCa, as well as for facilitating prognosis assessment and treatment evaluation. With ongoing technological advancements and further research, we believe that more methylation biomarkers will be approved and applied in clinical practice, providing additional benefits to patients with BCa.

4.1. Bladder EpiCheck

The bladder EpiCheck (BE) test (Nucleix, Rehovot, Israel) is a DNA methylation assay based on 15 genomic loci. Food and Drug Administration 510(k) clearance was obtained for the detection of BCa recurrence. This test involves centrifugation and extraction of genomic DNA from ≥10 mL of urine. The extracted DNA is then digested using methylation-sensitive restriction endonucleases that cleave unmethylated DNA while preserving the integrity of the methylated sequences. Subsequently, the digested DNA is amplified using real-time PCR with specific primers and probes, and the obtained data are analyzed using the BE software. This approach provides a reliable tool for screening for BCa recurrence.

Witjes et al.^[57] conducted a study involving 440 patients with suspected BCa, wherein their urine samples were collected prior to cystoscopy. Among them, 353 patients who met the standard criteria for histopathologically confirmed recurrent or incident urothelial carcinoma and underwent regular cystoscopic monitoring every 3 months were included in the cohort. The researchers utilized the BE technology to analyze urine samples and compared the results with follow-up outcomes, with the goal of evaluating the sensitivity and specificity of BE in detecting recurrent BCa. The findings revealed that, during the follow-up of patients with NMIBC, the BE test demonstrated an impressive overall negative predictive value (NPV) of 95.1%, with a remarkable 99.3% NPV for excluding the recurrence of LG Ta tumors. Furthermore, the BE exhibited a high specificity of 88.0%. The clinical application of the BE can potentially reduce the frequency of subsequent cystoscopy examinations, thereby alleviating the burden on patients and reducing health care costs.^[57] Witjes et al.^[57] demonstrated that the sensitivity of the BE varies depending on the stage and grade, a result consistent with other research related to BE. Moreover, the sensitivity was higher in higher stages (Ta, T1, and carcinoma in situ: 100%, 100%, and 1%, respectively) and higher-grade tumors (40% for LG and 89% for high-grade tumors).^[58] Notably, other DNA methylation markers exhibited similar characteristics.^[38,59] Pierconti et al.^[60] conducted a study involving 151 patients with high-grade NMIBC to investigate the diagnostic accuracy of BE during the follow-up of patients with highly malignant NMIBC, comparing it with concurrent urine cytology examination. The results showed that the overall specificity rates of the BE and urine cytology were similar at 85.1% and 86.3%, respectively. However, in the group of patients with papillary high-grade NMIBC, the BE demonstrated a higher specificity than cytology examination (96.3% vs. 90.4%). Throughout all follow-up periods for papillary NMIBC and carcinoma in situ, the BE consistently exhibited higher sensitivity than cytological examinations. Multiple research findings strongly suggest that the BE holds promise as a valuable noninvasive adjunctive diagnostic tool for the follow-up of patients with NMIBC. Compared with conventional urine cytology, BE has the potential to integrate and surpass, exhibiting superior performance in clinical practice.^[57,59–63]

4.2. URIFIND

URIFIND, an early BCa detection product based on urine DNA methylation testing, has received Breakthrough Device Designation from the US FDA, making it the first of its kind in China to receive such recognition. Building upon the utMeMA model developed by Chen et al.,^[36] URIFIND exhibited a sensitivity of 87.8% and specificity of 85.4%, resulting in an overall accuracy of 86.6%. Notably, in the validation group, URIFIND demonstrated a high positive predictive value of 96.0% (with 80% prevalence of BCa), providing robust support for the clinical diagnosis of BCa. This breakthrough further affirms the innovative and significant role of URIFIND in early screening of BCa, offering the potential for more reliable and efficient detection methods in clinical practice. Its deployment is expected to positively affect early diagnosis and treatment of BCa by equipping clinicians with a dependable and efficient tool.

4.3. Human TWIST1

We conducted a thorough search on PubMed and found numerous studies exploring the association between the TWIST1 gene and BCa.^{[40,47,53–65]} The earliest study utilizing real-time fluorescence qPCR to detect TWIST1 methylation levels in monitoring BCa was conducted by Reinert et al.^[64] In a study conducted by Hermanns et al.,^[47] the qPCR-MethyLight method was used to examine urine samples from 211 patients with BCa, including 180 individuals with NMIBC and 102 healthy individuals. The results demonstrated significant hypermethylation of 6 biomarkers (p < 0.0001), with sensitivity ranging from 82% to 89% and specificity ranging from 94% to 100%. These findings highlight the potential of TWIST1 methylation detection using real-time fluorescence qPCR as a promising approach for BCa monitoring. Identification of these methylation markers may offer valuable insights into the early detection and risk assessment in patients with BCa.^[64] Hermanns et al.^[47] used qPCR-MethyLight method to examine the urine samples of 211 patients with BCa, including 180 individuals with NMIBC and 102 healthy individuals. The results of the experiment revealed that Twist1 exhibited a sensitivity of 77.6% and specificity of 61.1% for predicting the presence of BCa and its grading. These findings suggest that TWIST1 detection using qPCR-MethyLight method is a promising diagnostic tool for BCa. However, further research is required to enhance its accuracy as well as specificity and sensitivity for broader clinical applications.^[47] Recently, a testing kit utilizing the Twist1 gene as a methylation biomarker was approved by the NMPA in China and has become a new method for the adjunctive diagnosis of BCa. Multiple clinical studies conducted in China have demonstrated that this testing kit exhibits a high sensitivity of 88.08% and specificity of 90.48%, with an overall concordance rate of 89.64%. This kit provides a reliable and effective adjunctive diagnostic tool for patients with BCa, with the potential to facilitate early diagnosis and treatment and improve patient prognosis. However, despite these encouraging results, further research and clinical validation are needed to ensure the robustness and accuracy in a broader population.

5. Advancements in DNA methylation detection techniques

Despite the notable advancements and breakthroughs in urine DNA methylation testing technology in recent years, inherent limitations and challenges remain. Traditional methods such as methylation-specific restriction enzyme digestion, although easy to perform, require prior knowledge of the restriction enzyme cut sites of the target genome and do not provide high-resolution methylation information. Similarly, MSP allows for qualitative detection of methylation status but has limited quantitative capability to accurately measure methylation levels. Furthermore, the use of bisulfite treatment in DNA analysis can result in DNA degradation, further complicating the process.^[34] However, these conventional methods have limitations that hinder their extensive clinical use. These limitations include the need for large sample quantities of DNA, lengthy experimental durations, complex operational procedures, and inherent instability of measurement outcomes.^[66]

To overcome these limitations, researchers are actively exploring new technologies to improve the existing DNA methylation detection processes. Through a comprehensive analysis of recent literature in the field of “urine DNA methylation detection” and “liquid biopsy,” we identified the potential of microfluidic technology,^[42–44] digital droplet PCR (ddPCR),^[67] CRISPR, and other emerging technologies in the field of methylation detection. These advances hold great promise for advancing DNA methylation detection and open new possibilities in the field.

5.1. Microfluidic technology

Microfluidics involves the manipulation and control of fluids at the microscale level, typically within channels ranging from micrometers to nanometers in size. It is also known by several other names, such as lab-on-a-chip technology, micro–total analytical system, microfluidic-based analysis, and microfluidic chip technology. Researchers are actively exploring the application of microfluidics technology in DNA methylation detection and analysis. This technology can miniaturize at least one application into the chip format, thereby reducing the overall analysis time and improving the efficiency and reliability. An example is the system developed by Keeley et al.,^[42] in which a single-tube extraction and processing technique known as “bead-based methylation” allows for the extraction of DNA and bisulfite conversion from 2 mL of liquid samples, such as plasma. This innovative method enhances the extraction efficiency and enables the conversion of DNA into bisulfite, facilitating subsequent methylation analysis. Moreover, it mitigates the presence of PCR inhibitors that often persist with traditional DNA extraction methods, thus enabling the detection of rare epigenetic events and facilitating high-sensitivity epigenetic diagnostic analysis. Microfluidic systems offer the advantages of integrating multiple steps and automating the entire analysis process, thereby reducing the risk of contamination and minimizing sample consumption.^[68] Yoon et al.^[43] developed a laboratory system called LoMA-B, which integrates bisulfite conversion with on-chip sample preprocessing and label-free real-time analysis modules. This system utilizes isothermal DNA amplification and detection technology to rapidly analyze the DNA methylation status. Compared with traditional MS-PCR, the LoMA-B system can analyze the methylation status of genomic DNA within 80 minutes, whereas traditional MS-PCR requires 24 hours. Moreover, the LoMA-B system demonstrates high sensitivity, enabling the detection of methylated DNA at concentrations as low as 1% in a mixture of methylated and nonmethylated cells. As a result, the LoMA-B system is regarded as a straightforward, versatile, and quantifiable diagnostic tool for evaluating DNA methylation patterns, with promising prospects for clinical applications. Lee et al.^[44] developed an innovative device for DNA methylation-specific amplification and detection that combined microfluidic systems with a flexible plastic-based on-chip nucleotide digestion device. This device incorporates optimized magnetic field effects and methylation-specific isothermal solid-phase amplification and detection technologies. It aims to enable the real-time, simple, and cost-effective analysis of DNA methylation in cancer-related DNA biomarkers. The device demonstrated rapid detection of methylated genes in the genomic DNA of human cancer cell lines within 65 minutes, surpassing the speed and accuracy of other methods. This groundbreaking technology holds great potential for clinical applications, offering an effective solution for fast and accurate analysis of DNA methylation.^[44] Microfluidic technology is an emerging field that has shown remarkable performance and promising prospects for DNA methylation detection. Its high-throughput capability, automation, and heightened sensitivity offer researchers increased options and opportunities. Although the current emphasis of many studies has not revolved around detecting DNA methylation levels in urine for BCa diagnosis, we believe that microfluidic technology has significant potential in this domain.

5.2. Digital droplet PCR

Digital droplet PCR is an innovative technique that integrates microfluidics with digital PCR.^[69] By partitioning PCR reactions into individual droplets and leveraging the principles of Poisson statistics, ddPCR allows for the accurate and precise quantification of target nucleic acids in a sample.^[70] Digital droplet PCR offers several distinct advantages over real-time qPCR. Digital droplet PCR allows absolute quantification without the need for external reference standard curves.^[71–73] This eliminates the reliance on calibration curves and enhances the accuracy of quantification. Moreover, through target DNA/RNA enrichment, ddPCR achieved remarkable sensitivity at the level of individual nucleic acid molecules. This enhanced sensitivity opens up the possibility of detecting rare genetic variants and low-abundance targets. Additionally, ddPCR demonstrates robustness in the presence of PCR inhibitors, making it applicable for the absolute quantification of DNA in challenging sample types such as animal blood, feces, urine, sputum, and other complex biological matrices.^[71] Digital droplet PCR has extensive applications in diverse fields, including the detection of rare mutations, copy number variations, and gene rearrangements. Although its use in DNA methylation analysis remains limited, its distinctive advantages highlight its enormous potential. With continued advancements and refinements in ddPCR technology, ddPCR has emerged as a pivotal tool for DNA methylation research. By offering enhanced sensitivity and precision, ddPCR opens new avenues for unraveling the intricate role of DNA methylation in disease onset and progression.^[74–78] Pharo et al.^[46] performed comprehensive methylation profiling of BCa cell lines and identified 8 promising biomarkers. These biomarkers were integrated into a detection panel called BladMetrix. The performance of BladMetrix was evaluated using ddPCR in an independent, prospective, blinded urine series of 273 patients with visible hematuria. The results revealed a remarkable sensitivity (92.1%), specificity (93.3%), and a high NPV (98.1%) for BladMetrix. These findings highlight the potential of BladMetrix to significantly reduce the frequency of cystoscopic examinations by 56.4%, making it a valuable tool for noninvasive BCa detection.^[46] Although research in this area remains relatively limited, ddPCR holds great promise for detecting DNA methylation in urine samples, surpassing the limitations of real-time qPCR. Owing to its numerous advantages, including higher sensitivity, improved quantification, and reduced interference from PCR inhibitors, ddPCR is expected to find increasing applications in tumor screening, specifically in the field of BCa. As technology continues to advance and its utility expands, we anticipate that ddPCR will provide clinicians with more accurate and reliable tools for the clinical diagnosis and prognostic analysis of BCa.

5.3. CRISPR

CRISPR technology is a revolutionary technique for genome editing and modification inspired by the natural immune systems of bacteria and archaea. By harnessing the collaborative action of Cas proteins and guiding RNA within the CRISPR-Cas system, CRISPR technology enables the precise editing and modification of target DNA sequences.^[79] The CRISPR technology demonstrates remarkable sensitivity, specificity, and simplicity in nucleic acid detection, thus holding immense potential for clinical diagnostics and capturing broad interest. Although clinical studies investigating the application of CRISPR technology for the detection of urine DNA methylation in BCa diagnosis are yet to be published, its diverse features make it a promising avenue. Researchers have validated the significance of CRISPR technology in methylation detection. Li et al.^[80] introduced a novel approach, HOLMESv2, which utilizes the thermostable CRISPR-Cas12b system for the precise quantification of DNA methylation levels. By combining Cas12b detection with bisulfite treatment, HOLMESv2 offers an accurate and efficient method for assessing the degree of methylation of the target DNA. Li et al.^[80] applied the HOLMESv2 method to measure methylation levels in 4 different cell lines treated with bisulfite and compared the results with established techniques, such as clone-based bisulfite sequencing, bisulfite sequencing PCR direct sequencing, and next-generation sequencing-based methylation sequencing. Remarkably, the HOLMESv2 method showed a high level of agreement with next-generation sequencing, which is considered the criterion standard for quantifying DNA methylation. These findings underscore the potential of HOLMESv2 as a powerful platform for molecular diagnostics and epigenetics research, highlighting its promise in advancing our understanding of DNA methylation and its role in various biological processes.^[80]

6. Discussion

Despite the several studies that have contributed to a multitude of DNA methylation detection methods, the clinical implementation of these technologies remains challenging. For example, numerous studies on BE have shown that, under similar conditions, BE exhibits higher sensitivity, positive predictive value, and NPV than urinary cytology. However, its relatively high cost puts it at a disadvantage compared with the current clinical criterion standard for diagnosing NMIBC, which is the TURBT/flexible cystoscopy.

In reality, as existing technologies continue to mature and new methods are being applied, BE and other methylation detection techniques are continually improving in diagnostic efficacy while also achieving lower costs. Furthermore, we believe that the true advantage of these methylation detection methods over TURBT/flexible cystoscopy lies in the prognostic staging of NMIBC. Non–muscle-invasive BCa is characterized by a propensity for recurrence, requiring patients to undergo regular flexible cystoscopic examinations, which can be inconvenient and uncomfortable. In this context, noninvasive methods, such as BE, with their high sensitivity, can play a pivotal role.

7. Conclusions

This comprehensive review extensively explores the application of DNA methylation in BCa detection and emphasizes its significance as a potential diagnostic biomarker. We specifically highlight several FDA/NMPA-approved methylation detection kits and their crucial role in practical applications. These certified kits are reliable tools for BCa methylation detection and have been widely adopted.

Furthermore, we introduce emerging technologies and methods for overcoming the limitations of traditional approaches. Microfluidics, ddPCR, and CRISPR are some of the novel techniques that have been discussed. Microfluidics offers automated and integrated analyses that reduce operational risks and sample consumption. Digital droplet PCR, with its advantages of absolute quantification and resistance to PCR inhibitors, has shown promise for methylation detection. CRISPR technology enables the precise editing and modification of target DNA sequences, thereby facilitating accurate methylation analyses.

In summary, we are confident in future research and the application of these advancements. Although further studies and validations are necessary in the field of methylation detection, these new technologies provide more accurate, efficient, and reliable methods for BCa methylation analysis and provide clinicians with reliable diagnostic and prognostic tools. The convergence of genomics, epigenetics, and bioinformatics will continue to promote the development of methylation biomarkers. Through ongoing efforts and collaborative endeavors, we strive to deliver increasingly precise and effective methylation analysis tools, thereby driving advancements in the field of medicine.