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Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer

Kitchen, Mark O.; Bryan, Richard T.; Haworth, Kim E.; Emes, Richard D.; Luscombe, Christopher; Gommersall, Lyndon; Cheng, K. K.; Zeegers, Maurice P.; James, Nicholas D.; Devall, Adam J.; Fryer, Anthony A.; Farrell, William E.


Mark O. Kitchen

Richard T. Bryan

Kim E. Haworth

Professor of Bioinformatics

Christopher Luscombe

Lyndon Gommersall

K. K. Cheng

Maurice P. Zeegers

Nicholas D. James

Adam J. Devall

Anthony A. Fryer

William E. Farrell


Bing-Hua Jiang



Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.


Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.


The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).


In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.


Kitchen, M. O., Bryan, R. T., Haworth, K. E., Emes, R. D., Luscombe, C., Gommersall, L., …Farrell, W. E. (2015). Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer. PLoS ONE, 10(9), e0137003.

Journal Article Type Article
Acceptance Date Aug 11, 2015
Online Publication Date Sep 2, 2015
Publication Date Sep 2, 2015
Deposit Date Dec 18, 2015
Publicly Available Date Dec 18, 2015
Journal PLoS ONE
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 10
Issue 9
Article Number e0137003
Pages e0137003
Public URL
Publisher URL
Copyright Statement Copyright information regarding this work can be found at the following address:


journal.pone.0137003.pdf (635 Kb)

Copyright Statement
Copyright information regarding this work can be found at the following address:

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