A methylation assay for the detection of non‐muscle‐invasive bladder cancer (NMIBC) recurrences in voided urine

What's known on the subject? and What does the study add? Multiple studies report on the detection of methylation in voided urine samples as a possible approach for the follow‐up of non‐muscle invasive bladder cancer patients. Previous studies analyze methylation gene panels in a mixture of primary and recurrent tumours. As primary tumours are larger than recurrent tumours and thus easier to detect in urine, validation of methylation markers in urine samples from patients with primary tumours will result in a test sensitivity that does not reflect the true sensitivity of the assay. This study is the first to select a subset of genes specifically methylated in non‐muscle invasive bladder cancer recurrences and validates the gene panel in two independent sets of urine samples from recurrent patients, thus simulating the disease course according to the clinical presentation. OBJECTIVE To develop a methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) assay for the detection of non‐muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS Genes frequently methylated in NMIBC tumours ( n = 37) were selected to develop a BC‐specific MS‐MLPA assay. Genes methylated in blood from patientswith BC ( n = 29) and genes methylated in urine from patients with no history of BC ( n = 46) were excluded. A four‐gene panel with the highest predictive value was selected from the initial assay. This four‐gene panel was tested and validated on urine from patients with a histologically confirmed recurrence ( n = 68 test set; n = 49 validation set) and urine samples from patients without BC ( n = 91, test set) and urine from recurrence‐free BC (rec‐free BC) patients ( n = 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four‐gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples ( n = 65) and on urine samples from rec‐free BC patients ( n = 29). RESULTS The BC MS‐MLPA assay consisted of 23 methylation probes. The selected four‐gene panel included: APC_a , TERT_a , TERT_b , and EDNRB . This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.