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


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom; Urology Department, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom.
Mark O
MO
Kitchen
School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom.
Richard T
RT
Bryan
Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom.
Kim E
KE
Haworth
Advanced Data Analysis Centre, University of Nottingham, Nottingham, United Kingdom.
Richard D
RD
Emes
Urology Department, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom.
Christopher
C
Luscombe
Urology Department, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom.
Lyndon
L
Gommersall
School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom.
K K
KK
Cheng
Department of Complex Genetics, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands.
Maurice P
MP
Zeegers
Cancer Research Unit, University of Warwick, Coventry, United Kingdom.
Nicholas D
ND
James
School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom.
Adam J
AJ
Devall
Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom.
Anthony A
AA
Fryer
Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom.
William E
WE
Farrell
D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor; D019175:DNA Methylation; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000235:genetics*; D060787:Neoplasm Grading; D011379:Prognosis; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology*
D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D014157:Transcription Factors; C097899:homeobox protein HOXA9; C063843:insulin gene enhancer binding protein Isl-1