Research Report

Protocadherin-17 promoter methylation in serumderived DNA is associated with poor prognosis of bladder cancer

Journal of International Medical Research 2014, Vol. 42(1) 35–41 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513504705 imr.sagepub.com

Zhen-Guo Luo1, Zhi-Gang Li2, Shi-Liang Gui1, Bao-Jin Chi1 and Jian-Guo Ma3

Abstract Objective: To investigate the prognostic value of protocadherin 17 (PCDH17) promoter methylation in serum-derived DNA of patients with bladder cancer. Methods: DNA was isolated from serum of patients with bladder cancer and from age- and sexmatched controls. Methylation-specific polymerase chain reaction was used to examine the methylation status of the PCDH17 promoter. The correlations between methylation status and clinicopathological characteristics and overall survival were examined. Results: PCDH17 promoter methylation was detected in 79/151 (52.3%) of patients with bladder cancer, and none of the 43 control subjects. Methylation was significantly associated with larger tumour diameter (>3 cm), high grade (G3) and advanced stage (T2–T4). Patients with PCDH17 promoter methylation had significantly shorter overall survival than those with unmethylated PCDH17 promoter. Methylation was an independent predictor of overall survival. Conclusions: PCDH17 promoter methylation was significantly associated with malignant behaviour and poor prognosis of bladder cancer. The detection of PCDH17 promoter methylation in serum-derived DNA may be a convenient and noninvasive predictive biomarker in routine clinical practice.

Keywords Biomarker, protocadherin-17, methylation, bladder cancer, prognosis Date received: 4 August 2013; accepted: 18 August 2013

1

Department of Urology, First Hospital of Jiamusi University, Jiamusi, Heilongjiang Province, China 2 Department of Urology, The General Hospital of CNPC in Jilin, Jilin, Jilin Province, China 3 Department of Urology, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China

Corresponding author: Jian-Guo Ma, Department of Urology, Third Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China. Email: [email protected]

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Introduction Bladder cancer is the second most common genitourinary neoplasm, with an estimated 72 570 new cases and 15 210 deaths expected to occur in the USA in 2013.1 Due to the aging population, the incidence of bladder cancer morbidity is likely to increase in the future and to remain a global healthcare problem.2 The initial presentation is superficial in 80% of cases, but superficial bladder cancer recurs in 70% of patients in spite of treatment standardization, with 10–30% of these patients developing invasive carcinoma.3 Survival is negatively correlated with tumour progression.3 Bladder cancers are histopathologically, morphologically and behaviourally heterogeneous, and it is therefore important to identify which tumours are likely to progress and which patients will need aggressive postoperative adjuvant therapy to improve their prognosis.4 Conventional prognostic factors such as tumour stage and grade are clinically useful, but their accuracy remains unsatisfactory.5 Novel, precise prognostic methods are therefore required. The initiation and progression of bladder cancer is characterized by the gradual accumulation of genetic and epigenetic changes that lead to the activation of proto-oncogenes or inactivation of tumour suppressor genes.6 The main epigenetic modification in human cancers is DNA methylation.7 This is a potential prognostic biomarker, especially in cases where the aberrant methylation inactivates tumour suppressor genes.8 DNA methylation can be detected in both tumour tissue-derived DNA and cell-free circulating DNA in serum.9 Methylation of circulating DNA can usually be detected if the tumour DNA is methylated, since these DNA fragments show the same characteristics as the primary tumour DNA.10 Analysis of DNA methylation in serum is sensitive, noninvasive, relatively cheap, and suitable for routine clinical use.11

Journal of International Medical Research 42(1) The protocadherin 17 (PCDH17) gene is located on chromosome 13q21.2 in humans, and functions as a tumour suppressor. Its TATA-less promoter contains CG-rich sequences that are susceptible to DNA methylation. PCDH17 methylation is found in several human carcinomas, including bladder, gastric and colorectal cancers.12–14 Methylation-specific polymerase chain reaction (MSP) is a simple, sensitive, specific and cost-effective method for detection of DNA methylation.11 The present study used MSP to investigate PCDH17 promoter methylation in serum samples from patients with bladder cancer, in order to evaluate its prognostic significance.

Patients and methods Study population The study recruited patients with primary bladder cancer undergoing treatment at the Department of Urology, Third Hospital of Hebei Medical University, Shijiazhuang, China, between May 2003 and May 2008. Inclusion criteria were: histopathological diagnosis of transitional cell carcinoma of the bladder; no anticancer treatment before blood sampling; no other prior tumour; availability of complete follow-up data. Tumour diagnosis, staging, therapy and follow-up were completed according to international standards.15 Overall survival was defined as the time from the date of diagnosis to the date of death from any cause, or last contact if the patient was still alive. Age- and sex-matched control subjects were recruited from healthy individuals with no history of any tumour, who were attending the same hospital for routine health checks. Written informed consent was obtained from each participant. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics

Luo et al. Committee of the Third Hospital of Hebei Medical University.

Methylation-specific PCR Peripheral blood samples were obtained from all the participants before any therapy using standard methods as described previously.16 Serum was isolated and stored at 80 C until use. DNA was extracted from 500 ml of serum using a QIAampÕ DNA Blood mini kit (Qiagen, Valencia, CA, USA) then treated with bisulphite to convert any unmethylated (but not methylated) cytosine residues to uracil (EpiTect Bisulfite Kit; Qiagen). PCDH17 methylation status was examined using MSP as described previously.12 Primer sequences were: PCDH17 unmethylated sense, 50 -AGATTATTGGGTGTTGTA 0 GTTT-3 and antisense, 50 -AACCCTAA CACAACATACACA-30 (90-base pair product); and PCDH17 methylated sense, 50 -GATTATCGGGTGTCGTAGTTC-30 and antisense, 50 -CCCTAACGCAACGTA CGCG-30 (87-base pair product). The PCR cycling conditions were as follows: denaturation at 95 C for 5 min, followed by 40 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 1 min and extension at 72 C for 1 min, followed by a final extension step at 72 C for 5 min. In vitro methylated DNA and unmethylated DNA (Millipore, Billerica, MA USA) were used as positive controls for methylation and unmethylation, respectively. Water was used as a negative control in each assay. PCR products were separated in 2% agarose gels, stained with ethidium bromide, and visualized under ultraviolet illumination. Samples were scored as methylation positive when methylated alleles were present in the methylated DNA lane and methylation negative when bands were present only in the unmethylated DNA lane.16 PCR was performed a minimum of three times per sample.

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Statistical analyses Data were presented as n (%). Fisher’s exact test was used to evaluate the difference in PCDH17 methylation status between patients and controls, and 2-test was used to assess the relationship between PCDH17 methylation and clinicopathological parameters. Kaplan–Meier survival analysis and log-rank test were used to assess the difference in overall survival between methylated and unmethylated PCDH17. Multivariate Cox proportional hazard analysis was used to estimate the independent prognostic effect of PCDH17 methylation, controlling for classic risk factors (tumour size, stage and grade).17–19 Statistical analyses were carried out using SAS version 8.0 (SAS Institute, Cary, NC, USA). P-values 65 Sex Male Female No. of tumours Single Multiple Tumour diameter, cm 3 >3 Tumour shape Papillary Nonpapillary Grade G1/G2 G3 Stage Ta–T1 T2–T4

151

72 (47.7)

79 (52.3)

55 96

30 (54.6) 42 (43.8)

25 (45.5) 54 (56.3)

NS

107 44

49 (45.8) 23 (52.3)

58 (54.2) 21 (47.7)

NS

58 93

32 (55.2) 40 (43.0)

26 (44.8) 53 (57.0)

NS

88 63

50 (56.8) 22 (34.9)

38 (43.2) 41 (65.1)

P ¼ 0.0079

102 49

50 (49.0) 22 (44.9)

52 (51.0) 27 (55.1)

NS

98 53

56 (57.1) 16 (30.2)

42 (42.9) 37 (69.8)

P ¼ 0.0016

92 59

54 (58.7) 18 (30.5)

38 (41.3) 41 (69.5)

P ¼ 0.0007

Data presented as n (%). a 2  -test.

survival than those with methylated PCDH17 (P < 0.0001; Figure 2). Cox proportional hazard analysis revealed that PCDH17 promoter methylation status, tumour stage, tumour grade and tumour diameter were independent predictors of overall survival (Table 2).

Discussion DNA methylation is common in human cancers and plays a critical role in the regulation of gene expression, differentiation and tumour development.20 DNA methylation occurs mainly in cytosine–guanine

Luo et al.

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Figure 2. Kaplan–Meier survival analysis of patients with bladder cancer (n ¼ 151), stratified according to protocadherin 17 (PCDH17) promoter methylation status assessed in serum-derived DNA. P < 0.0001; logrank test.

Table 2. Multivariate Cox proportional hazard analysis of independent predictors of overall survival in patients with bladder cancer (n ¼ 151), controlling for classic risk factors (tumour size, stage and grade). Variable

Hazard ratio

95% confidence interval

PCDH17 promoter methylation status, methylated vs unmethylated Stage, T2 – T4 vs Ta – T1 Grade, G3 vs G1/G2 Tumour diameter, >3 cm vs 3 cm No. of tumours, multiple vs single

4.758

1.871, 11.127

3.582 2.941 2.547 2.241

1.685, 1.213, 1.043, 0.905,

10.237 8.117 7.615 6.178

PCDH17, protocadherin-17; HR, hazard ratio.

dinucleotide-rich areas (CpG islands) in gene promoter regions.20 Promoter methylation may be used as a biomarker in cancer risk evaluation, diagnosis and prognosis, and in determining sensitivity to chemo- or radiotherapy.21–24 DNA methylation can be detected in body fluids as well as tumour tissue, since double-stranded DNA fragments are present in the serum of patients with cancer,9,11,25,26 and this DNA has a similar methylation status to that found in tumours.4 Circulating tumour DNA may derive from tumour cells or result from

DNA leakage caused by tumour necrosis or apoptosis.9,20 The current study investigated the relationship between PCDH17 promoter methylation detected in serumderived DNA and clinicopathological characteristics and overall survival of patients with bladder cancer. It has been shown that PCDH17 functions as a tumour suppressor and is downregulated by promoter methylation in human cancers.12,13 PCDH17 promoter methylation is common in urological cancers including bladder cancer, renal cell

40 carcinoma and prostate cancer.14 PCDH17 promoter methylation was detected in serum-derived DNA of 79/151 (52.3%) of patients but none of the control subjects in the present study, indicating that it is tumour-specific. In the current study, two cases were shown as being both unmethylated and methylated, which can be termed partial methylation. It is well known that serum-derived DNA originates from tumour tissue DNA; DNA methylation is a gradual process in which some DNA is methylated and some is not, thus resulting in partial methylation. In addition, PCDH17 methylation was significantly associated with larger tumour size, high grade and advanced stage in the current study, all of which are classic risk factors for unfavourable outcomes in bladder cancer.27,28 We found that patients with methylated PCDH17 had significantly shorter overall survival than those with unmethylated PCDH17, and PCDH17 methylation was an independent prognostic indicator. These findings suggest that PCDH17 promoter methylation detected in serum-derived DNA is a potential prognostic biomarker in bladder cancer. This study was limited by its small sample size. In addition, we did not use a positive control (a tissue-specific gene). Further larger-scale studies are required to confirm our findings. In conclusion, this study found that PCDH17 promoter methylation was significantly associated with malignant behaviour and poor prognosis of bladder cancer. The detection of PCDH17 promoter methylation in serum-derived DNA may be a convenient and noninvasive predictive biomarker in routine clinical practice. Patients with PCDH17 promoter methylation may require systemic adjuvant therapy after initial surgery in order to achieve better outcomes. Declaration of conflicting interest The authors declare that there is no conflict of interest.

Journal of International Medical Research 42(1) Funding This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.

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