HumanMethylation450K Array-Identified Biomarkers Predict Tumour Recurrence/Progression at Initial Diagnosis of High-risk Non-muscle Invasive Bladder Cancer

Abstract

Background: High-risk non-muscle invasive bladder cancer (HR-NMIBC) is a clinically unpredictable disease. Despite clinical risk estimation tools, many patients are undertreated with intra-vesical therapies alone, whereas others may be over-treated with early radical surgery. Molecular biomarkers, particularly DNA methylation, have been reported as predictive of tumour/patient outcomes in numerous solid organ and haematologic malignancies; however, there are few reports in HR-NMIBC and none using genome-wide array assessment. We therefore sought to identify novel DNA methylation markers of HR-NMIBC clinical outcomes that might predict tumour behaviour at initial diagnosis and help guide patient management.

Patients and methods: A total of 21 primary initial diagnosis HR-NMIBC tumours were analysed by Illumina HumanMethylation450 BeadChip arrays and subsequently bisulphite Pyrosequencing. In all, 7 had not recurred at 1 year after resection and 14 had recurred and/or progressed despite intra-vesical BCG. A further independent cohort of 32 HR-NMIBC tumours (17 no recurrence and 15 recurrence and/or progression despite BCG) were also assessed by bisulphite Pyrosequencing.

Results: Array analyses identified 206 CpG loci that segregated non-recurrent HR-NMIBC tumours from clinically more aggressive recurrence/progression tumours. Hypermethylation of CpG cg11850659 and hypomethylation of CpG cg01149192 in combination predicted HR-NMIBC recurrence and/or progression within 1 year of diagnosis with 83% sensitivity, 79% specificity, and 83% positive and 79% negative predictive values.

Conclusions: This is the first genome-wide DNA methylation analysis of a unique HR-NMIBC tumour cohort encompassing known 1-year clinical outcomes. Our analyses identified potential novel epigenetic markers that could help guide individual patient management in this clinically unpredictable disease.

Keywords: HumanMethylation450 BeadChip array; epigenetics; high-risk non-muscle invasive bladder cancer; methylation; prognostic biomarker.

Figure 1.

Heatmap of the 206 differentially methylated CpG sites between the clinical outcomes of HR-NMIBC. Heatmap of the differentially methylated CpG sites identified by array analysis. The heatmap separates the 14 recurrence or progression tumours on the left (n = 14) from the no-recurrence tumours on the right (n = 7). Each row represents an individual CpG locus, and each column represents a tumour sample (listed beneath the heatmap). The colour scale beneath the heatmap represents methylation status: unmethylated is blue (β value = 0.0) and fully methylated is red (β value = 1.0).

Figure 2.

Receiver operating characteristic (ROC) curves for cg11850659 and cg01149192. ROC curves for the 2 best performing biomarker candidates. Hypermethylation of CG11850659 (left) – AUC: 0.71 (95% CI: 0.57-0.83) and hypomethylation of CG01149192 (right) – 0.64 (95% CI: 0.50-0.77). AUC indicates area under the curve; CI, confidence interval.