Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation.
BACKGROUND
Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine.


RESULTS
The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%-89% and specificity in the range 94%-100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88-94% and specificities of 43-67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P ≤ 0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR=7.8, P=0.006), POU4F2 (HR=8.5, P=0.001), TWIST1 (HR=12.0, P=0.015), VIM (HR=8.0, P=0.001), and ZNF154 (HR=13.9, P<0.001). Interestingly, for one group of patients (n=15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect.


CONCLUSIONS
Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
Thomas
T
Reinert
Michael
M
Borre
Anders
A
Christiansen
Gregers G
GG
Hermann
Torben F
TF
Ørntoft
Lars
L
Dyrskjøt
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000652:urine; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics / Q000652:urine; D006801:Humans; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009364:Neoplasm Recurrence, Local; D009687:Nuclear Proteins / Q000235:genetics / Q000652:urine; D011379:Prognosis; D020825:T-Box Domain Proteins / Q000235:genetics / Q000652:urine; D050820:Transcription Factor Brn-3B / Q000235:genetics / Q000652:urine; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000652:urine; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology / Q000652:urine; D014746:Vimentin / Q000235:genetics / Q000652:urine; D016335:Zinc Fingers / Q000235:genetics
D014408:Biomarkers, Tumor; C491199:EOMES protein, human; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C494190:POU4F2 protein, human; D020825:T-Box Domain Proteins; C103173:TWIST1 protein, human; D050820:Transcription Factor Brn-3B; D051879:Twist-Related Protein 1; D014746:Vimentin; C097899:homeobox protein HOXA9