Hypothesis / aims of study
Literature on the prevalence of bladder cancer (BCa) in patients with NLUTD is conflicting. Large studies have reported a similar incidence for BCa in catheterized individuals with spinal cord injury (SCI) to that observed in the general population (see review [1]), albeit with a considerably higher risk for muscle-invasive BCa at presentation. Other studies report 16–28 times higher risk of BCa in NLUTD individuals compared to the general population, with an incidence as high as 10% [2]. In another patient series screened with urethrocystoscopy and bladder wash cytology, a range of relevant histological findings were found in 5% of them [1]. Bladder wash cytology and/or urethrocystoscopy are recommended by EAU bladder cancer guidelines, spinal cord medicine guidelines and the International Consultation on Incontinence as part of routine follow-up for NLUTD patients [1]. Although urethrocystoscopy, urine cytology and/or bladder biopsies have been used for the screening of NLUTD patients for BCa, no established screening protocol exists. Moreover, some researchers support the idea to avoid screening of all NLUTD patients due to the low incidence of the disease, low effectiveness and high cost of the screening methods. Therefore, the development of urine biomarkers for BCa seems an attractive non-invasive method of screening in this patient population.
DNA methylation is an epigenetic modification, occurring frequently in genes’ promoters and resulting in transcriptional suppression. DNA methylation has been found in cancerous tissues as a mechanism of inactivation of tumor suppression genes. In BCa is a frequent event found in 50-90% of cases. DNA hypermethylation was also found in normal appearing urothelium in patients with BCa supporting further the hypothesis that this event occurs in early stages of bladder tumors’ pathogenesis. Nowadays, DNA hypermethylation with the application of modern methods can, also, be detected in urine samples with high sensitivity and specificity.
The aim of this study is to evaluate, for the first time, the presence of DNA methylation of a panel of five genes’ promoters in the urine of patients with NLUTD, and in comparison with previously reported results from normal individuals
Study design, materials and methods
This is a pilot, prospective, observational study conducted in the setting of a Neurourology outpatient clinic. The study protocol was approved by the Hospital Review Board and all patients signed written informed consent. Eligible participants had a diagnosis of neurological disease affecting the lower urinary tract with NLUTD duration of at least five years. Exclusion criteria were age under 18 years, prior diagnosis and treatment of BCa and prior diagnosis of another urinary tract malignancy.
DNA Methylation process. For study purposes, 50 ml of urine were collected via spontaneous urination, intermittent self-catheterization or indwelling catheter, depending on patients’ voiding habits, supplemented with urine preservative (Urine Preservative Single Dose, Norgen Biotek Corp) and stored at room temperature. DNA was extracted from urine sediments using the Cells and Tissue DNA Isolation Kit (Norgen Biotek Corp) and modified with sodium bisulfite employing the EZ DNA Methylation-GoldKit (Zymo Research), according to the manufacturer's instructions. Quantitative Methylation Specific PCR (qMSP) was performed to detect bisulfite-induced changes at the promoter of the following gene: RASSF1, RARβ, DAPK, hTERT and APC. These genes, alone or in combination panels, were found to be hypermethylated in the urine of BCa patients and suggested to be risks factors in meta-analyses.
qMSP was performed with Luna Universal Probe qPCR Master Mix (New England Biolabs, USA), using the Applied Biosystems StepOnePlus Real Time PCR System (Thermo Fisher Scientific, Inc.).
Statistical analyses were performed using the statistical package R (Version 3.4.4 for Windows) and R studio (Version 1.1.453 for Windows). Results from this patient cohort were compared to those previously reported on the same panel of genes in normal controls [3]. Chi-squared was used to compare the results
Results
33 subjects with NLUTD were recruited over a period of time from October of 2018 to March of 2019. The baseline characteristics of the NLUTD group are shown in Table 1. DNA was detected in 28 of 33 urine samples. DNA was found to be hypermethylated in at least one of five gene promoters in 11 urine samples (39.29%). DNA hypermethylation was found in one gene in 8 samples (27.58%), in two genes in 2 samples (6.9%), in three genes in 1 sample (3.45%), while DNA hypermethylation in four or five gene promoters was found in no sample (Figure 1). RASSF1 was hypermethylated in 6/11 samples (54.55%) with detected methylation, APC in 5/11 samples (45.45%), DAPK in 3/11 samples (27.59%), RAR-β2 in 1/11 sample (9.09%) and hTERT in no sample.
Characteristics of the normal control group (n=35) have been previously reported [3]. DNA was detected in 22 of 35 control samples. DNA was found to be hypermethylated in 3 samples (13.64%), all in RASSF1 gene promoter.
When comparing the two groups, DNA methylation was more frequently detected in the NLUTD patients than in the control group (11 (39.29%) vs 3 (13.64%), p=0.045).
Interpretation of results
Results from this mixed etiology NLUTD patient cohort could indicate a higher risk for BCa in NLUTD patients, as meta-analyses have shown that the hypermethylation of RASSF1, DAPK and APC promoters in urine is associated with higher BCa risk. Previous studies had suggested a higher risk for BCA in NLUTD patients with indwelling catheters, but our study population comprised mostly patients emptying with clean intermittent catheterizations (CIC), suggesting that the risk is independent of the type of catheter used for emptying. The majority of patients had a smoking history and a history of recurrent UTIs, but our study sample is rather small for robust conclusions on the negative prognostic value of any of these confounding factors during screening for BCa.
This is the first study to evaluate DNA methylation in the urine as biomarker for BCa in this specific population, while a previous study analyzed the efficacy of cytology, BTA stat test and survinin in a cohort of SCI patients with negative results. So, further assessment and surveillance of our cohort is needed to prove the possible association of our findings with BCa.