Urinary NID2 and TWIST1 methylation to augment conventional urine cytology for the detection of bladder cancer.

BACKGROUND: Abnormal methylation of urinary TWIST1 and NID2 conferred high sensitivity and specificity for the detection of urothelial carcinoma. OBJECTIVE: We examine the performance of the urine-based TWIST1/NID2 methylation assay with the addition of urine cytology for the detection of urothelial carcinoma. MATERIALS AND METHODS: A prospective multi-institutional study was conducted to assess the performance of a methylation assay for patients with hematuria or under surveillance for non-muscle invasive bladder cancer (NMIBC). All patients underwent cystoscopy, a methylation assay, and cytology. Receiver operator characteristic (ROC) curves were constructed for cytology alone, the methylation assay alone, and a combined model. Areas under the curve (AUC) were compared using likelihood ratio tests. RESULTS: A total of 172 patients were enrolled (37% for hematuria and 63% NMIBC). The AUC for cytology alone with equivocal cytologies positive was 0.704, and improved to 0.773 with the addition of the DNA methylation assay (p < 0.001). When the equivocal cytologies were considered negative, the AUC improved from 0.558 to 0.697 with the addition of the DNA methylation assay (p = 0.003). CONCLUSIONS: Addition of a TWIST1/NID2-based DNA methylation assay adds diagnostic value to urine cytology and the model is sensitive to the classification of equivocal cytology.

Multi-institutional external validation of urinary TWIST1 and NID2 methylation as a diagnostic test for bladder cancer.

OBJECTIVES: We previously reported a clinical trial in which we were unable to replicate the excellent diagnostic metrics produced in the developmental study of the TWIST1 and NID2 gene methylation assay. In this expanded trial with subjects enrolled from another institution, we reexamine the diagnostic capabilities of the test to externally validate our previous study. MATERIALS AND METHODS: TWIST1 and NID2 gene methylation was assessed in DNA isolated from the urine of subjects at risk of bladder cancer undergoing cystoscopy for hematuria or bladder cancer surveillance. The diagnostic gold standard was cystoscopy. Two thresholds of TWIST1 and NID2 gene methylation were used for determining test result positivity, those published by Renard et al. and Abern et al. The sensitivity, specificity, positive and negative predictive values, diagnostic likelihood ratios, and receiver operating characteristic curves were calculated for each gene, as well as their combination. In all, 3 methods were used to combine TWIST1 and NID2 into a single composite test: (1) believe-the-positive decision rule-if either gene is methylated the test result is positive, which maximizes test sensitivity; (2) believe-the-negative decision rule-if either gene is not methylated the test result is negative, which maximizes test specificity; and (3) a likelihood-based logistic regression model approach that balances sensitivity and specificity. Clinical utility was determined using a decision curve analysis. RESULTS: A total of 209 subjects were evaluated: 40% for hematuria and 60% for bladder cancer surveillance. Approximately 75% were male, most of the prior cancers being low-grade Ta. Using cystoscopy as the gold standard, areas under the curve were 0.67 for TWIST1, 0.64 for NID2, and 0.66 for combined TWIST1 and NID2. Decision rule results revealed optimization of sensitivity at 67% using Renard thresholds and specificity using the Abern thresholds at 69%. We found improved sensitivity (78%) in current smokers. Decision curve analyses revealed that the methylation assay provided only a modest benefit even at high probabilities of missed cancer. CONCLUSION: A urine DNA test measuring TWIST1 and NID2 methylation was externally examined with a larger cohort and its results continue to be poor. These 2 biomarkers are unlikely to replace cystoscopy, but they may be worthy of study in active smokers.

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.

A noninvasive multianalyte urine-based diagnostic assay for urothelial cancer of the bladder in the evaluation of hematuria.

OBJECTIVE: To test whether a noninvasive urine-based multianalyte diagnostic readout assay that uses protein and DNA biomarkers can risk stratify patients with hematuria into those who are or are not likely to have bladder cancer and those who should receive standard care. PATIENTS AND METHODS: This prospective, observational, multicenter, single-assessment study was conducted between June 12, 2009, and April 15, 2011. Eligible patients presented with hematuria and as part of their evaluation underwent cystoscopy. Urine samples were analyzed for the presence of mutant FGFR3 and quantified matrix metalloproteinase 2 and the hypermethylation of TWIST1 and NID2. A patient's chance of having (positive predictive value [PPV]) or not having (negative predictive value [NPV]) cancer was determined by FGFR3 alone or by all 4 biomarkers, respectively. RESULTS: Cystoscopy/biopsy diagnosed 690 of 748 patients as negative and 58 as positive for bladder cancer. Of 21 patients identified by FGFR3 as highly likely to have cancer, 20 were also positive by cystoscopy/biopsy, resulting in a PPV of 95.2% (20 of 21), with specificity of 99.9% (689 of 690). The 4-marker combination identified 395 patients as having a low likelihood of cancer. Of these, 56.2% (388 of 690) also had negative biopsy/cystoscopy findings, resulting in an NPV of 98.2% (388 of 395). In total, 416 of the 748 patients with hematuria (55.6%) were identified with extremely high NPV and PPV to have or not have bladder cancer. CONCLUSION: This multianalyte assay accurately stratified patients with high confidence into those who likely do or do not have bladder cancer. This test was developed to enhance and not to eliminate referrals for urologic evaluation.