Improved urine DNA methylation panel for early bladder cancer detection.

BACKGROUND: Bladder cancer is one of the most common malignancies but the corresponding diagnostic methods are either invasive or limited in specificity and/or sensitivity. This study aimed to develop a urine-based methylation panel for bladder cancer detection by improving published panels and validate performance of the new panel with clinical samples. METHODS: Related researches were reviewed and 19 potential panels were selected. RRBS was performed on a cohort with 45 samples to reassess these panels and a new panel inherited best markers was developed. The new panel was applied with qMSP platform to 33 samples from the RRBS cohort and the results were compared to those of RRBS. Lastly, another larger cohort with 207 samples was used to validate new panel performance with qMSP. RESULTS: Three biomarkers (PCDH17, POU4F2 and PENK) were selected to construct a new panel P3. P3 panel achieved 100% specificity and 71% sensitivity with RRBS in corresponding cohort and then showed a better performance of 100% specificity and 84% sensitivity with qMSP platforms in a balanced cohort. When validated with 207-sample cohort, P3 with qMSP showed a performance of 97% specificity and 87% sensitivity which was modestly improved compared to the panels it derided from. CONCLUSIONS: Overall, the P3 panel achieved relatively high sensitivity and accuracy in bladder cancer detection.

HOXA9, PCDH17, POU4F2, and ONECUT2 as a Urinary Biomarker Combination for the Detection of Bladder Cancer in Chinese Patients with Hematuria.

BACKGROUND: DNA methylation biomarkers for bladder cancer (BCa) have not been evaluated extensively in the Chinese population. OBJECTIVE: To develop and validate a urinary biomarker combination of methylation assays in a group of Chinese patients with hematuria. DESIGN, SETTING, AND PARTICIPANTS: A total of 192 urine samples were collected and evaluated from patients with microscopic or gross hematuria, including 97 BCa patients and 95 controls with benign diseases. A two-stage study was conducted: the first stage being assay construction and the second stage being assay validation. Eighty-one urine samples were analyzed for the hypermethylation of eight selected genes in stage 1 and then a four-gene panel was constructed. An additional 111 urine samples were analyzed using the four-gene panel (including HOXA9, PCDH17, POU4F2, and ONECUT2) for independent validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The positive predictive value (PPV) and negative predictive value (NPV) were calculated for the combination methylation assay. Uni- and multivariate binary logistic regression analyses (backward elimination, conditional) were performed to calculate the association between BCa and each predictor variable. RESULTS AND LIMITATIONS: The combination assay of HOXA9, PCDH17, POU4F2, and ONECUT2 was selected based on the results of multivariate logistic regression analysis in stage 1. Using a strategy of three-level risk stratification, the assay yielded a consistent PPV of 100%. With an estimated BCa prevalence of 10% in a general hematuria population, the assay would result in an overall NPV of 98%. This combined methylation biomarker would yield an overall area under the receiver operating characteristic curve of 0.871 (with a sensitivity of 90.5% and a specificity of 73.2%) if using the prediction model from multivariate regression analysis. In addition, over half of BCa cases would be predicted accurately and ∼60% of unnecessary cystoscopies could be spared. This study had several limitations. First, the sample size was relatively small. Second, it was performed in a case-control population rather than in a natural hematuria cohort. CONCLUSIONS: A combination methylation assay of HOXA9, PCDH17, POU4F2, and ONECUT2 resulted in high PPV and NPV in Chinese patients with hematuria. With accurate risk prediction, the urinary biomarker combination could spare a sizeable proportion of low-risk patients from extensive and invasive examination. PATIENT SUMMARY: In the present study, we looked at the predictive performance of a urinary biomarker combination of HOXA9, PCDH17, POU4F2, and ONECUT2. We found that this urinary biomarker combination may help discriminate bladder cancer from other benign diseases in patients with hematuria, resulting in a reduction of unnecessary invasive examination in patients at low risk.

Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation.

BACKGROUND: Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine. METHODOLOGY/PRINCIPAL FINDINGS: The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%-89% and specificity in the range 94%-100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88-94% and specificities of 43-67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P ≤ 0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR=7.8, P=0.006), POU4F2 (HR=8.5, P=0.001), TWIST1 (HR=12.0, P=0.015), VIM (HR=8.0, P=0.001), and ZNF154 (HR=13.9, P<0.001). Interestingly, for one group of patients (n=15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect. CONCLUSION/SIGNIFICANCE: Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.