Urine DNA Methylation Detection for Hematuria Evaluation

Methods

Study Design:

This is a prospective pilot study aimed at evaluating the effectiveness of DNA methylation biomarkers in detecting urothelial carcinoma in patients with hematuria.

Sample Size Calculation:

Based on our previous observations, the incidence of urothelial carcinoma in patients with hematuria is 20%-30%, slightly higher than reported in other literature. Therefore, we hypothesize that 25% of the patients in the hematuria cohort have UC. The group allocation ratio (R): N-/N+ = 75%/25% = 3.

Assumptions:

Area under the curve (AUC) under H0: 0.5 AUC under H1: 0.8 Power: 0.95 Significance level (Alpha): 0.05 Type of data: Continuous FPR range: 0.00 to 1.00

Results: Using PASS 15.0 software for sample size calculation to ensure sufficient statistical power. The calculated sample size is:

N+ (number of patients with UC): 14 N- (number of patients without UC): 42 Total sample size (N): 56

Considering a 20% dropout rate, the adjusted sample size is:

N+': 18 N-': 53 Total sample size (N'): 71

Inclusion and Exclusion Criteria:

Inclusion Criteria:

Aged between 18 and 99 years, with gross or microscopic hematuria (>3/HP). Able to provide 50ml urine for testing before surgery. Consent to participate in the study and sign the informed consent form.

Exclusion Criteria:

With history of malignancy or concomitant malignancies other than UC. Severe urinary tract infection leading to sepsis. Patients with indwelling catheters, nephrostomy, or cystostomy. Severe liver or kidney failure or other conditions deemed unsuitable for the study.

Patients who did not undergo surgical treatment for various reasons. Samples with insufficient DNA content or other quality control failures.

Sample Collection:

Clinicians will collect fresh urine samples from enrolled hematuria patients and record their basic information, clinical information, and medical history.

Samples will be randomly numbered and provided to DNA methylation testing personnel to ensure blinding.

Testing Method:

Urine samples were collected in EP Genomic DNA Kit with an automated nucleic acid extraction instrument. Subsequently, 100 ng of genomic DNA was used for methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) detection as described previously. Different from bisulfite PCR relying on bisulfite conversion, MSRE-qPCR is based on the selective digestion of DNA by methylation-sensitive enzyme followed by qPCR with primers that surround the cutting site.

Unblinding and Data Organization:

After the last sample is successfully enrolled and tested, non-recruiting personnel and testing personnel will unblind the samples and organize clinical and pathological information.

Data Analysis:

Using pathological results as the gold standard. Calculate the sensitivity, specificity, positive predictive value, and negative predictive value of DNA methylation biomarkers.

Use statistical methods (such as ROC curves) to evaluate the diagnostic performance of DNA methylation biomarkers and calculate the AUROC.

Ethics and Informed Consent:

This study has been approved by the hospital's ethics committee, and all participants must sign an informed consent form.