Diagnosis of Bladder Cancer Recurrence Based on Urinary Levels of <em>EOMES</em>, <em>HOXA9</em>, <em>POU4F2</em>, <em>TWIST1</em>, <em>VIM,</em> and <em>ZNF154</em> Hypermethylation

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>The methylation levels of <em>EOMES</em>, <em>HOXA9</em>, <em>POU4F2</em>, <em>TWIST1</em>, <em>VIM,</em> and <em>ZNF154</em> 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%. <em>EOMES</em>, <em>POU4F2</em>, <em>VIM</em> and <em>ZNF154</em> 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; <em>HOXA9</em> (HR = 7.8, P = 0.006), <em>POU4F2</em> (HR = 8.5, P = 0.001), <em>TWIST1</em> (HR = 12.0, P = 0.015), <em>VIM</em> (HR = 8.0, P = 0.001), and <em>ZNF154</em> (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.</p> <h3>Conclusion/Significance</h3><p>Methylation levels of <em>EOMES</em>, <em>HOXA9</em>, <em>POU4F2</em>, <em>TWIST1</em>, <em>VIM,</em> and <em>ZNF154</em> in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.</p> </div

Demographic and clinical characteristics of bladder cancer patients and control individuals.

a<p>N/A Not available.</p>b<p>Bergkvist.</p>c<p>Of the 184 patients, 26 were lost for follow-up.</p>d<p>The presence of tumor cells in the urine was determined by urine cytology.</p><p>Demographic and clinical characteristics of bladder cancer patients and control individuals from whom urine specimens were collected and methylation analysis performed. Histology was used as the gold standard for the diagnosis of bladder tumors.</p

Follow-up model for low-risk NMIBC applying methylation markers.

<p>Modified from Hermann GG et al. (<a href="http://skejby.net/Webudgaven/DaBlaCa2010.htm" target="_blank">http://skejby.net/Webudgaven/DaBlaCa2010.htm</a>.).</p

Kaplan-Meier plot of the time to recurrence.

<p>DNA methylation is associated with subsequent tumor recurrence within 24 months for patients without tumor but with methylation-positive urine samples. Kaplan-Meier plots of recurrence-free survival as a function of dichotomized methylation levels for <i>EOMES</i> (P = 0.0397) (A), <i>HOXA9</i> (P = 0.0009) (B), <i>POU4F2</i> (P<0.0001) (C), <i>TWIST1</i> (P = 0.0017) (D), <i>VIM</i> (P = 0.0001) (E), and <i>ZNF154</i> (P<0.0001) (F).</p

Diagnostic significance of the urinary markers.

a<p>Some urine samples provided inconclusive results for some markers.</p>b<p>Positive predictive value.</p>c<p>Negative predictive value.</p>d<p>Mann-Whitney <i>U</i> test.</p>e<p>Not available.</p><p>Diagnostic significance of the urinary markers <i>EOMES</i>, <i>HOXA9</i>, <i>POU4F2</i>, <i>TWIST1, VIM,</i> and <i>ZNF154</i>, when comparing urine samples from 184 patients with NMIBC to urine from healthy individuals.</p

Diagnostic significance of the urinary markers for surveillance of bladder cancer when including a 12-months follow-up period.

a<p>Some urine samples provided inconclusive results for some markers.</p>b<p>Mann-Whitney <i>U</i> test.</p><p>Diagnostic significance of the urinary markers <i>EOMES</i>, <i>HOXA9</i>, <i>POU4F2</i>, <i>TWIST1, VIM,</i> and <i>ZNF154</i>, when comparing urine samples from patients with NMIBC to urine samples from bladder cancer patients with no recurrence and using DNA collected at control visits in patients with a methylation positive first tumor. Tumors diagnosed during a 12 month follow-up period were included. Histology was used as the gold standard for the diagnosis of bladder tumors.</p

Immunoblottings of right ventricle samples from normoxic and hypoxic rats.

<p>Proteins participating in metabolism A: ACAA2, B: HADHA and C: aquaporin 7 show tendency to downregulation by hypoxia. D: monoamine oxidase A, E: tissue transglutaminase and F: endothelin receptor B were all upregulated by hypoxia. ACAA2: acetyl-Coenzyme A acyltransferase 2, HADHA: hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit. Values are means ± SE and are calculated as percent of normoxia 1 week. n = 6 in both groups at all time points. *P<0.05 vs. normoxia at same time point.</p

Representative immunoblots for proteins measured in the right ventricle from normoxic and hypoxic rats.

<p>ACAA2: acetyl-Coenzyme A acyltransferase 2, HADHA: hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit.</p

Parameters describing the pulmonary and systemic impact of hypoxia.

<p>SSBP: Systemic systolic blood pressure; HW/BW: Heart weight to body weight ratio.</p><p>n = 6 in both groups at all time points. Values are means ± SE. *P<0.05 vs. normoxia at same time point.</p

Experimental design.

<p>*Three animals from each control and four animals from each hypoxic subgroup were used for GeneChip analysis. Four animals from each group either pulmonary trunk banded (PTB) or sham at week 5 were used for GeneChip analysis.</p