Meta-analysis of the Association Between APC Promoter Methylation and Bladder Cancer

Bladder cancer (BC) is a worldwide disease that affects a large number of people. This study analyzes the sensitivity and specicity of adenomatous polyposis coli (APC) methylation for BC detection in urine and tissues. Combining search results from PubMed and Embase, 20 studies were included. In tissue subgroups, the OR was 6.88 (95% condence interval (CI 2.49–19.03, P<0.001), respectively. From urine studies, the pooled OR was 4.73 at a 5% signicance level (95% CI 0.87-25.66, P<0.001). In addition, analysis of the interaction between APC methylation and BC showed strong association in the whole data set. From this study, the results suggest that APC promoter methylation may be the potential testing for BC diagnosis and provide a new viewpoint in the treatment of BC.


Introduction
Bladder cancer (BC) is one of the most prevalent type of urothelial cell carcinoma in industrialized countries, with papillary urothelial carcinoma of transitional cell origin as the predominant histologic type, and its incidence shows a yearly increasing trend 1 . Five-year overall survival rates in patients with invasive BC depend on the tumor in ltration depth. Although super cial bladder cancer generally has a good long-term prognosis, up to 80 % of patients will have local recurrence within 5 years of the primary tumor resection, which necessitates life-long cystoscopic follow-up and frequent transurethral resections Additionally, we hand-searched the references of the review articles and, as needed, contacted the rst author of a given paper to obtain any missing data.

Study selection
Studies were selected for the current meta-analysis according to the following criteria: they determined APC promoter methylation in specimens of bladder tissue, blood, plasma, serum, buffy coat, or urine; and they provided su cient information to evaluate odds ratios (ORs) and 95% con dence intervals (CIs). In addition, when the same author or group reported results that were obtained from the same patient population in more than one article, only the most recent or most informative report was included. Our exclusion criteria were: review articles or conference reports; a lack of information about the degree of APC promoter methylation in patient cases and controls; and when screening for methylation of the APC promoter was conducted in cell lines.

Quality assessment
The assessment of study quality was conducted independently by two reviewers using the Newcastle-Ottawa Scale (NOS). The NOS evaluation system consists of three parameters (selection, comparability, and outcome) and assigns a maximum of four points for selection, two points for comparability, and three points for outcome. A NOS score > 6 indicates a higher quality study, whereas a score ≤ 6 indicates a lower quality study. Any discrepancies between the two reviewers were settled by a third reviewer.

Data extraction
The data were extracted independently by two authors, and discrepancies were resolved by consensus including a third author. The data were collected using a pilot-tested data extraction form that included the following items: the rst author's name; the year of publication; the number of participants that exhibited APC promoter methylation both among speci c cases and controls; the screening methods used; and the demographic and clinical characteristics of the patients. All procedures conformed to the established guidelines for the meta-analysis of observational studies in epidemiology.

Statistical analysis
The meta-analysis was performed using Stata software (version 12; StataCorp LP, College Station, TX, USA). The association between APC promoter methylation and the risk of developing BC or its clinical characteristics (such as in tissue ,in urine, as well as differences, recurrence or primary, based on patient sex) was measured either by weighted OR by taking into account the 95% CI. We tested for heterogeneity among the studies using the chi-square-based Q-test and the I2statistic of inconsistency. Signi cant heterogeneity was de ned as a chi-square test P-value < 0.10 or as an I2 statistic > 50%. We used a random-effects model when signi cant heterogeneity was observed among the studies; otherwise, we used a xed-effects model. For a two-tailed signi cance level of 5%, the probability of rejecting the null hypothesis when it was false was termed the power, which was de ned as 1 -β. Funnel plots and Egger's test were used to assess publication bias. Additionally, prespeci ed subgroup analyses, which included the patients' ethnicities, as well as the test samples, testing methods, and sample sizes, were conducted to evaluate potential sources of heterogeneity within the studies investigating the association between APC promoter methylation and the risk of BC. Moreover, sensitivity analyses were performed to examine the in uence of each study on the pooled OR by serially omitting each individual study and pooling the remaining studies.
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Results of the literature search
Illustrates the detailed process used for study selection. In summary, a total of 225 articles were initially identi ed; of these, 200 articles were excluded, either based on duplicate results or because they were deemed to be irrelevant to this meta-analysis after careful review of the titles and abstracts. Among the 25 studies that remained, an additional 5 articles were excluded for various reasons: 1 were review articles; 2 were discarded because screening of APC promoter methylation was performed using cell lines; and 2 were excluded because of a lack of data on the association between APC promoter methylation and the development of BC. Thus, 20 articles were ultimately selected for inclusion in the meta-analysis.

Study characteristics
The characteristics of the studies that met all of the established inclusion criteria for the meta-analysis are presented in Table 1. 9 studies used bladder tissue specimens to screen for APC promoter methylation, while 8 studies used urine.The numbers of articles that reported the associations between APC promoter methylation and sex of the BC patients was 2. For the detection of APC promoter methylation, 18 studies used methylation-speci c polymerase chain reaction, 2used quantitative real-time methylation-speci c polymerase chain reaction (Q-MSP).

Data quality
We estimated the quality of the studies using the NOS evaluation system, and the results showed that 15 of the 19 studies were classi ed as high-quality (NOS score > 6) and the remaining four trials were classi ed as lower-quality. The mean NOS score of the studies was 7. Most of the studies did not use community controls when we conducted an assessment of comparability.

Methylation of the APC promoter and BC
As shown in Fig. 2, 9 studies comprising 511 patients evaluated the association between APC promoter methylation and BC in tissue, (pooled OR 6.88; 95% CI 2.49-19.03, P < 0.001). 8 studies assessed the association between APC promoter methylation and BC in urine, and the pooled OR was 4.73 at a 5% signi cance level (95% CI 0.87-25.66, P < 0.001; Fig. 3). 2 studies assessed the association between APC promoter methylation and recurrence or primary BC, and the pooled OR was 1.44 at a 5% signi cance level (95% CI 0.25-8.17, P < 0.05). We also evaluated the association between APC promoter methylation and patient sex, and no signi cant association was identi ed between APC promoter methylation and patient sex ( Table 2).

Discussion
Currently, the gold standard for bladder cancer detection remains to be cystoscopy followed by histological examination for making the initial diagnosis and monitoring progression of bladder tumors. Although this approach provides valuable prognostic information regarding tumor status, it involves an invasive procedure that causes considerable discomfort to the patients which shows insu cient power to predict precisely the patient outcome and 10-40% of malignancies may be undetected. Because of the risk of recurrence, bladder cancer is a disease that requires life-long surveillance with periodic cystoscopy. Alternatively, urine cytology which has been used for decades is the standard noninvasive method for cancer detection. But the sensitivity of cytology is speci cally insu cient for the detection of low stage and grade bladder tumors, whereby its accuracy is in uenced by the pathologist's experience.
An array of urine markers have been identi ed to improve the diagnostic ability of urine cytology and, perhaps, reduce the number of cystoscopies, consequently improving patient quality of life and patient care. In view of this, there is a dire need to identify prognostic biomarkers that can serve as reliable indicators of disease and predict recurrence more accurately. Over the past two decades a number of biomarkers have been identi ed and approved by FDA for screening of bladder cancers but initial enthusiasm for their clinical utility waned quickly because of lacked speci city, reproducibility as well as sensitivity, especially for low grade and stage of bladder cancer 3 . Hence the clinical applicability of these markers remains limited. Therefore, a more sensitive and noninvasive method is imperative for e cient BC detection.
Epigenetic changes are de ned as changes in gene expression that are heritable through cell division, without associated DNA sequence alterations. DNA methylation occurs in distinct regions of promoters where cytosine residues located at the 5' position of guanines in CpG dinucleotides which is not randomly distributed but is especially important in CpG-rich areas found in over 60% of the human genes, also called CpG islands. Hypermethylation of promoter CpG islands and somatic mutations are among the most common and speci c types of genome alterations in human cancer and represent causative events in tumor development. Promoter hypermethylation negatively in uences transcription and mutations may lead to activation of proto-oncogenes or inactivation of tumor suppressor genes. Some epigenetic alterations occur early during tumorigenesis has been intensively investigated over the last ten years 4,5 , and could be used as targets for the molecular diagnosis of neoplastic cells in clinical specimens such as biological uids that are readily accessible. Aberrant DNA promoter hypermethylation has been described in bladder cancer and have shown promising results. It seems to be an early event in the development of a number of solid tumors including bladder cancer 6,7 and can thus be regarded as an early sign of cancer before the disease becomes muscle-invasive. The CpG methylation in BC has been reported to occur frequently in bladder cancer and to be associated with age, smoking status, gender, tumor location, stage, recurrence rate and progression 8-12 . In addition to detection of methylation in tumor tissue, analysis of gene methylation has been shown to be feasible from body uids, including voided urine of patients with BC and appears to be more sensitive than urine cytology.
In bladder cancer, a large number of genes have been shown to harbor promoter hypermethylation, including Adenomatous Polyposis Coli (APC) as one of the most consistent and frequent targets. Expression and function of APC is known to be impacted either by physical changes in the sequence of DNA or by unprogrammed DNA methylation 13,14 . It is isolated and mapped at chromosomal band 5q21 15,16 . Loss of APC function results in nuclear accumulation of β-catenin, which acts as a transcriptional activator, ultimately leading to loss of cellular growth control 17,18 . Many studies have reported a high methylation frequency of APC promoter region in bladder cancer [19][20][21] . And DNA hypermethylation at the APC promoter correlated with cancer speci c mortality following radical cystectomy.
Meta-analysis was used to evaluate the association between APC methylation and malignant tumors of bladder cancer, and to provide clues for further exploring its pathogenic effects and pathways in these diseases.

Data availability
All data generated or analyzed during the present study are included in this published article.

Contribution
Xuedong Chen and Peng Li conceived of the presented idea. Qi Wu developed the theory and performed the computations. Xuedong Chen and Qi Wu veri ed the analytical methods. Fuchen Xie encouraged Xuedong Chen to investigate and supervised the ndings of this work. All authors discussed the results and contributed to the nal manuscript.

Con ict of interest
No con icts exist for any speci ed author.   Meta-analysis of the association between APC promoter methylation and the risk of bladder cancer in tissue. The circles and horizontal lines correspond to the study speci c OR and 95% CI. The sizes of the data markers indicate the weight of each study in the analysis.