Urine cell based DNA methylation classifier for monitoring bladder cancer

Abstract

Background: Current standard methods used to detect and monitor bladder cancer (BC) are invasive or have low sensitivity. This study aimed to develop a urine methylation biomarker classifier for BC monitoring and validate this classifier in patients in follow-up for bladder cancer (PFBC). Methods: Voided urine samples (N = 725) from BC patients, controls, and PFBC were prospectively collected in four centers. Finally, 626 urine samples were available for analysis. DNA was extracted from the urinary cells and bisulfite modificated, and methylation status was analyzed using pyrosequencing. Cytology was available from a subset of patients (N = 399). In the discovery phase, seven selected genes from the literature (CDH13, CFTR, NID2, SALL3, TMEFF2, TWIST1, and VIM2) were studied in 111 BC and 57 control samples. This training set was used to develop a gene classifier by logistic regression and was validated in 458 PFBC samples (173 with recurrence). Results: A three-gene methylation classifier containing CFTR, SALL3, and TWIST1 was developed in the training set (AUC 0.874). The classifier achieved an AUC of 0.741 in the validation series. Cytology results were available for 308 samples from the validation set. Cytology achieved AUC 0.696 whereas the classifier in this subset of patients reached an AUC 0.768. Combining the methylation classifier with cytology results achieved an AUC 0.86 in the validation set, with a sensitivity of 96%, a specificity of 40%, and a positive and negative predictive value of 56 and 92%, respectively. Conclusions: The combination of the three-gene methylation classifier and cytology results has high sensitivity and high negative predictive value in a real clinical scenario (PFBC). The proposed classifier is a useful test for predicting BC recurrence and decrease the number of cystoscopies in the follow-up of BC patients. If only patients with a positive combined classifier result would be cystoscopied, 36% of all cystoscopies can be prevented.

DNA Methylation and Bladder Cancer: Where Genotype does not Predict Phenotype

“…(A higher resolution / colour version of this figure is available in the electronic copy of the article). markers in biological fluids (blood, urine) can act as a predictive marker up to certain extent [13,14]. However, considerable methylation differences in these pre-diagnostic tests already indicates that molecular damage has already been done, hence left no choice except treatment improvement for patients to prolong their survival.…”

DNA methylation biomarkers of future health outcomes in children

“…It can be difficult to obtain samples from the tissue of interest in living humans, and particularly children as they are less tolerant of invasive testing. Potential solutions involve using non-invasive sampling methods such as induced sputum [47] or urine samples [48] which have successfully been used for DNA methylation analysis [49,50]. Another potential method for analysing tissue-specific DNA methylation in a relatively non-invasive manner is by analysing cell-free DNA, which is made up of small fragments of DNA that circulate in the blood and are thought to originate from apoptotic/dying cells [51].…”

Tumoral markers in bladder cancer (Review)

“…Studies have shown that hypermethylated genes such as adenomatous polyposis coli (APC), glutathione S-transferase π1 (GSTP1) and retinoic acid receptor β2 (RARb2) are frequently found in the urine of urothelial carcinoma patients; thus, the methylation status of cyclin-dependent kinase inhibitor 2A (p16INK4A), death-associated protein kinase 1 (DAPK), ARF tumor suppressor (p14ARF), APC and Ras association domain family member 1 (RASSF1A) tumor-suppressor genes have been found to be associated with the stage and grade of bladder cancer (10)(11)(12). Other studies have evaluated twist family BHLH transcription factor 1 (TWIST1) and nidogen 2 (NID2) gene methylation as well as spalt like transcription factor 3 (SALL3) and cystic fibrosis transmembrane conductance regulator (CFTR) concluding that the combination with cytology increases both the negative predictive value and sensitivity in patients with urothelial carcinoma or studied DNA methylation patterns that help distinguish non-invasive from muscle-invasive bladder cancer (13,14). In addition, new studies concerning DNA methylation have recently provided evidence for superior prognostic value for bladder tumor recurrence compared with classic diagnostic tools, by analyzing SRY-box transcription factor 1 (SOX-1), interleukin-1 receptor associated kinase 3 (IRAK3) and Li-MET gene methylation grade.…”