The authors have declared that no competing interests exist.
Conceived and designed the experiments: TR MB TFØ LD. Performed the experiments: TR. Analyzed the data: TR AC GGH TFØ LD. Contributed reagents/materials/analysis tools: TR MB TFØ LD. Wrote the paper: TR TFØ LD.
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.
The methylation levels of
Methylation levels of
Cancer of the urinary bladder is the fifth most common neoplasm in the industrialized countries. In approximately 75% of all cases, the patients will present with stage Ta or T1 non-muscle invasive bladder cancer (NMIBC), whereas the remaining 25% of the tumors will be muscle invasive stage T2-4 cancers (MIBC)
Epigenetics is the study of mitotically and/or meiotically heritable changes in gene expression that cannot be explained by changes in DNA sequence
We now validated the diagnostic and prognostic value of these biomarkers together with earlier reported
Informed written consent was obtained from all patients, and research protocols were approved by the Central Denmark Region Committees on Biomedical Research Ethics.
A total of 652 voided urine samples were collected at the Department of Urology at Aarhus University Hospital from 390 bladder cancer patients and 47 individuals with benign prostatic hyperplasia or bladder stones, but no history of bladder cancer (control individuals). From these we excluded 227 samples, because the DNA amount was below our threshold (
Characteristics | Control individuals | ||
|
35 | ||
Gender, n (%) | |||
Male | 30 (86) | ||
Female | 5 (14) | ||
Age, mean (min-max) | 70 (35–88) | ||
Nitrite test, n (%) | |||
Positive | 15 (43) | ||
Negative | 20 (57) | ||
|
|
|
|
|
184 | 101 |
57 |
Samples collected | 184 | 139 | 67 |
Primary cases | 44 | ||
Recurrent cases | 140 | 139 | |
Gender, n (%) | |||
Male | 148 (81) | 106 (76) | 58 (87) |
Female | 36(19) | 33 (24) | 9 (13) |
Age, mean (min-max) | 69 (33–89) | 71 (43–89) | 69 (49–86) |
Ta | 69 (33–85) | 70 (43–87) | |
T1 | 70 (42–89) | 74 (43–89) | |
CIS | 71 (67–74) | 73 (66–81) | |
T2-4 | 0 | 71 (43–83) | |
Pathological stage, n (%) | |||
Ta | 132 (72) | 92 (66) | |
T1 | 50 (27) | 29 (21) | |
CIS | 2 (1) | 5 (4) | |
T2-4 | 0 | 13 (9) | |
Grade, n (%) |
|||
I | 17 (9) | 12 (9) | |
II | 74 (40) | 55 (40) | |
III | 93 (51) | 71 (51) | |
Nitrite test, n (%) | |||
Positive | 16 (9) | 13 (9) | 7 (10) |
Negative | 163 (89) | 121 (87) | 57 (85) |
N/A |
5 (3) | 5 (4) | 3 (5) |
Tumor cells in urine |
|||
Positive | 119 (65) | 87 (63) | 22 (33) |
Negative | 28 (15) | 25 (18) | 24 (36) |
N/A | 37 (20) | 27 (19) | 21 (31) |
N/A Not available.
Bergkvist.
Of the 184 patients, 26 were lost for follow-up.
The presence of tumor cells in the urine was determined by urine cytology.
Demographic and clinical characteristics of bladder cancer patients and control individuals from whom urine specimens were collected and methylation analysis performed. Histology was used as the gold standard for the diagnosis of bladder tumors.
DNA was extracted with the QIAsymphony Virus/Bacteria Midi kit (96) (Qiagen) using the QIAsymphony® SP instrument and employing the Complex800_V5_DSP protocol. Five hundred nanograms of DNA was bisulfite modified using EZ-96 DNA methylation D5004 (Zymo Research) according to the manufacturer’s recommendations and eluted in 60 µl of elution buffer and stored at −20°C until use.
Methylation analysis was performed using methyLight
Stata 11 (Statacorp, Texas, USA) was used for all statistical calculations. Two-tailed tests were considered statistically significant if P<0.05. Methylation differences were evaluated by nonparametric Wilcoxon-Mann-Whitney test. Fisher’s exact test was used for analyzing dichotomous variables. The exact $x^2 $test was used for analyzing associations between clinic-pathological parameters with two or more categories. Correlations of the methylation levels of the markers were calculated with Spearman correlation coefficients. A ROC curve was made for each marker and combinations of markers by plotting sensitivity against (1-specificity) and the area under the curve (AUC) was calculated. Log-Rank tests were applied to evaluate equality of survival and Kaplan-Meier survival plots were used for visualization. Univariate Cox regression analysis was used to analyze associations of age, gender, stage, grade, multiplicity, and CIS with recurrence-free survival.
Our analysis was divided into two parts: 1) to establish the cutoff level of the methylation markers and to demonstrate the significance of the selected markers we analyzed the first urine sample from each patient and compared to control urine samples from healthy individuals; 2) using the determined marker cutoff levels we then validated the diagnostic and prognostic value of the methylation markers in urine samples taken during patient surveillance.
Initially, we defined the cut-off levels by the mean methylation level of each marker +2x standard deviation of the methylation level in urine samples from 35 control individuals (only samples with methylation values above zero were included). Cut-off levels (PMR values – see
Gene | Sensitivity, % (pos./total |
Specificity, % (neg./total) | AUC (95% CI) | PPV |
NPV |
P value |
|
88 (160/182) | 97 (34/35) | 0.96 (0.94–0.99) | 99 | 61 |
|
|
82 (141/173) | 100 (35/35) | 0.91 (0.88–0.94) | 100 | 52 |
|
|
85 (154/182) | 94 (33/35) | 0.94 (0.91–0.97) | 99 | 54 |
|
|
88 (159/180) | 100 (35/35) | 0.94 (0.92–0.97) | 100 | 63 |
|
|
89 (159/179) | 100 (35/35) | 0.97 (0.94–0.99) | 100 | 64 |
|
|
87 (160/184) | 100 (35/35) | 0.95 (0.93–0.97) | 100 | 59 |
|
|
81 (119/147) | N/A |
N/A | 100 | N/A | N/A |
Some urine samples provided inconclusive results for some markers.
Positive predictive value.
Negative predictive value.
Mann-Whitney
Not available.
Diagnostic significance of the urinary markers
We validated the clinical usefulness of the markers for bladder cancer surveillance. We stratified our analysis to only include patients that initially showed hypermethylation of one or more methylation markers. Depending on the marker studied, 11–18% of patients showed no methylation in the first tumor and was therefore not included. This restricted the analysis to 158 patients and 206 urine samples from the follow-up visits; 139 urine samples were from patients with a recurrent bladder tumor and 67 urine samples were from patients with no tumor recurrence (
Gene | Sensitivity, % (pos./total |
Specificity, % (neg./total |
AUC (95% CI) | PPV, % | NPV, % | P value |
|
94 (116/124) | 39 (24/61) | 0.78 (0.71–0.85) | 76 | 75 |
|
|
92 (108/117) | 38 (18/48) | 0.70 (0.61–0.80) | 78 | 67 |
|
|
87 (104/120) | 47 (28/60) | 0.75 (0.68–0.83) | 76 | 64 |
|
|
89 (113/127) | 28 (17/60) | 0.71 (0.63–0.80) | 72 | 55 |
|
|
90 (113/126) | 43 (24/56) | 0.72 (0.63–0.81) | 78 | 65 |
|
|
93 (115/123) | 47 (29/62) | 0.78 (0.71–0.86) | 78 | 78 |
|
|
77 (88/115) | 60 (35/58) | 0.68 (0.61–0.76) | 79 | 56 |
|
Some urine samples provided inconclusive results for some markers.
Mann-Whitney
Diagnostic significance of the urinary markers
The molecular tests may have a higher sensitivity compared to the gold standard cystoscopy. To address this we therefore used cystoscopy results from a 12 months period after the urine was sampled. We found many of the samples formerly classified as false positives to be true positives; they simply had a positive lead time compared to cystoscopy. The sensitivity obtained ranged from 88% to 94%, and the specificity ranged from 43% to 67% (
Gene | Sensitivity, % (pos./total |
Specificity, % (neg./total |
AUC (95% CI) | PPV, % | NPV, % | P value |
|
94 (133/141) | 55 (24/44) | 0.85 (0.77–0.92) | 87 | 75 |
|
|
93 (123/132) | 55 (18/33) | 0.78 (0.68–0.89) | 89 | 67 |
|
|
88 (120/136) | 64 (28/44) | 0.80 (0.72–0.89) | 88 | 64 |
|
|
90 (133/147) | 43 (17/40) | 0.76 (0.66–0.86) | 85 | 55 |
|
|
90 (129/143) | 59 (23/39) | 0.78 (0.68–0.89) | 89 | 62 |
|
|
94 (134/142) | 67 (29/43) | 0.83 (0.74–0.92) | 91 | 78 |
|
|
79 (99/126) | 74 (35/47) | 0.77 (0.69–0.84) | 89 | 56 |
|
Some urine samples provided inconclusive results for some markers.
Mann-Whitney
Diagnostic significance of the urinary markers
To address the prognostic value of the methylation markers we analyzed the urine samples from patients at visits where no tumors were diagnosed using cystoscopy. For all markers we found that a positive marker at a tumor-negative visit was significantly associated with later tumor recurrence during 24- and 60-month follow-up periods (Log-Rank test, P≤0.04) (
DNA methylation is associated with subsequent tumor recurrence within 24 months for patients without tumor but with methylation-positive urine samples. Kaplan-Meier plots of recurrence-free survival as a function of dichotomized methylation levels for
If the methylation of the biomarkers was confined to malignant cells, we should only detect the markers in urine when a tumor was present, or occurring within a foreseeable future depending on the growth rate of the tumor. However, our results showed that even high urinary levels of methylation could be present at visits without recurrences (
Modified from Hermann GG et al. (
In this study we performed an independent validation of
The controls applied in this study were from patients with benign prostatic hyperplasia (BPH) or bladder stones and 43% of the controls were nitrite positive, indicating bladder infections. Some of the cells in the urine of the controls may originate from the hyperplasia, or may be immunological or bacterial cells. Similar control samples were applied when the markers were initially investigated as markers of bladder cancer, suggesting that the methylation frequency of the selected markers is low in these cell types
It is noteworthy that only 9% of the recurrences are grade I. Therefore the calculated sensitivities of the markers may be higher compared to sensitivities that would be obtained from a consecutive series of low-risk patients with a much higher number of grade I tumors.
One of the main challenges using urinary markers is getting sufficient tumor cells, and while MethyLight is a very sensitive method we still had to exclude 227 (35%) samples from the study due to insufficient amounts of DNA. We observed that if we included samples with less than 5 ng of DNA as template in the analysis, the sensitivity decreased, and for patients under surveillance the specificity increased. By excluding patients based on the amount of DNA extracted from the voided urine samples, we maintain the integrity of the MethyLight assay at the cost of introducing a bias where especially patients with stage Ta grade I tumors are excluded, as grade I lesions exfoliate the smallest number of cells
The methylation marker test may be complemented by
The fact that we find no associations between methylation and clinicopathologic variables when using only urine from patients with recurrent tumors is intriguing, but it may be that the lower sensitivity observed in low-grade tumors is not only caused by few exfoliated tumor cells in the urine, but rather that the hypermethylation is not present in the tumor or in the surrounding urothelium, and by stratifying for methylation at an earlier visit these patients are excluded. It is possible that patients with no methylation are to be considered as a distinct group of bladder tumors. Finally, the lack of association between methylation and clinic-pathologic variables may also be a consequence of the relatively small data set.
The methylation markers have specificity values in the range 43% to 67%, which is low considering that they all have between 94%–100% tumor specificity when compared to control individuals. An equivalent low specificity was observed in a study investigating the mRNA levels of hTERT, SENP1, PPP1CA, and MCM5 in urine obtained from patients during disease surveillance
The methylation observed in urine samples from cystoscopy-negative visits may have several sources, (i) a small tumor not detected by cystoscopy, (ii) residual tumor cells at site of the resection, or (iii) it may be a symptom of epigenetically changed urothelial cells present in the urothelium surrounding the tumor or at other places (field defect) that have no phenotype to distinguish them from normal urothelial cells – an epigenetic urothelial methylator phenotype
With the discovery of methylation markers with very high sensitivity, implementation of methylation markers in the surveillance of patients with low-grade NMIBC seems likely. At the time of diagnosis, the methylation level of each methylation marker has to be established before the marker can be applied for surveillance. In advance of the next control visit, the patient may supply a urine sample for the analysis, and three possible outcomes exist: i) if the test is positive the patient will have a cystoscopy done, and in 67 patients out of 100 a recurrent tumor will be found, whereas the remaining 33 patients will not have a tumor recurrence. This is not a major problem, as the false positive patients would have had a cystoscopy done in any case; ii) a negative test will allow the patients to skip the current cystoscopy. With the current performance of the analysis, 94 out of 100 BC patients with a tumor recurrence are correctly diagnosed and six patients are wrongly diagnosed as not having a tumor recurrence. This amount of false negatives is comparable with HAL-guided cystoscopy and better than white light cystoscopy, where 20 patients can be expected to be wrongly diagnosed as not having a tumor recurrence
In conclusion, by applying a very sensitive and semi-quantitative methodology for detecting bladder cancer recurrences and stratifying for methylation status of the initial tumor, we have shown that using a single marker (
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We are grateful to Margaret Gellett and Anette Stenderup for their technical assistance. We thank the staff at the Departments of Urology at Aarhus University Hospital for their skillful assistance.