Identification and validation of methylated PENK gene for early detection of bladder cancer using urine DNA

Abstract

Background: Early detection of bladder cancer (BCa) offers patients a favorable outcome and avoids the need for cystectomy. Development of an accurate and sensitive noninvasive BCa diagnostic test is imperative. DNA methylation is an early epigenetic event in the development of BCa. Certain specific aberrant methylations could serve as useful biomarkers. The aim of this study was to identify methylation biomarkers for early detection of BCa.

Methods: CpG methylation microarray analysis was conducted on primary tumors with varying stages (T1-T4) and paired nontumor tissues from nine BCa patients. Bisulfite-pyrosequencing was performed to confirm the methylation status of candidate genes in tissues and urine sediments (n = 51). Among them, PENK was selected as a potential candidate and validated using an independent set of 169 urine sediments (55 BCa, 25 benign urologic diseases, 8 other urologic cancers, and 81 healthy controls) with a quantitative methylation-specific real time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0.

Results: CpG methylation microarray analysis and stepwise validation by bisulfite-pyrosequencing for tissues and urine sediments supported aberrant methylation sites of the PENK gene as potential biomarkers for early detection of BCa. Clinical validation of the mePENK-qMSP test using urine sediment-DNA showed a sensitivity of 86.5% (95% CI: 71.2 - 95.5%), a specificity of 92.5% (95% CI: 85.7 - 96.7%), and an area under ROC of 0.920 (95% CI: 0.863 - 0.959) in detecting Ta high-grade and advanced tumor stages (T1-T4) of BCa patients. Sensitivities for Ta low-grade, Ta high-grade, T1 and T2-T4 were 55.6, 83.3, 88.5, and 100%, respectively. Methylation status of PENK was not correlated with sex, age or stage, while it was associated with the tumor grade of BCa.

Conclusions: In this study, we analyzed the comprehensive patterns of DNA methylation identified that PENK methylation possesses a high potential as a biomarker for urine-based early detection of BCa. Validation of PENK methylation confirms that it could significantly improve the noninvasive detection of BCa.

Keywords: Bladder cancer; Methylation; Noninvasive; PENK; Urine sediment.

Fig. 1

Assessment of methylation levels of nine candidates in bladder tissues by bisulfite-pyrosequencing. Methylation status was determined for nine genes in primary tumor and corresponding nontumor tissues used in CpG microarray analysis. MtI values are plotted from pyrosequencing results. Samples from the same patients are linked with a straight line. NT: Nontumor tissues, T: Tumor tissues. *, P < 0.05 and **, P < 0.01 analyzed by paired t-test

Fig. 2

Methylation assessment of three genes in DNA from urine sediments by bisulfite-pyrosequencing. MtIs of samples are presented by box and whisker plots. Differences in MtI are statistically analyzed between BCa patients, BUD patients, and normal healthy subjects (N). *, P < 0.05 and **, P < 0.01 analyzed by Kruskal–Wallis test

Fig. 3

Methylation status of PENK in urine sediments by mePENK-qMSP. A mePENK-qMSP using an independent set of voided urine samples from BCa patients, BUD patients, and normal healthy subjects (N). Distribution of PENK methylation was expressed as 40-△C_T value for each sample. A higher 40-△C_T indicates a higher methylated level of PENK. Methylation status of PENK is plotted as box and whisker plots. TaLG: Ta Low-Grade; TaHG: Ta High-Grade; O.C.: Other urologic cancers. **, P < 0.01 analyzed by Kruskal–Wallis test. B ROC plots of PENK methylation for detecting TaHG and advanced tumor stages of BCa from BUD patients and normal healthy subjects. Cutoff value for methylation-positive, AUC, and P value are indicated in the box