- Top of page
- Material and Methods
- Supporting Information
DNA methylation patterns are associated with the development and prognosis of cancer. The aim of this study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation and RNA expression patterns in samples from long-term follow-up patients with nonmuscle invasive bladder cancer (NMIBC). A total of 187 human bladder specimens were used for microarray array or pyrosequencing (PSQ) analyses: 6 normal controls (NC) and 181 NMIBC. Tumor-specific hypermethylated genes were selected from a data set comprising 24 matched microarray-based DNA methylation and gene expression profiles (6 controls and 18 NMIBC), and their clinical relevance was verified by quantitative PSQ analysis. The methylation status of Homeobox A9 (HOXA9), ISL LIM homeobox 1 (ISL1) and Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) was significantly associated with decreased gene expression levels and aggressive clinicopathological characteristics. Multivariate regression analyses showed that hypermethylation of these genes was an independent predictor of disease recurrence (HOXA9, ISL1 and ALDH1A3, either alone or in combination) and progression (ISL1 and ALDH1A3, either alone or in combination) (each p < 0.05). The results of this study suggest that these novel methylation markers are independent prognostic indicators in NMIBC patients, which may facilitate the assessment of disease recurrence and progression in NMIBC patients and inform clinical decision making regarding treatment.
Nonmuscle invasive bladder cancer (NMIBC) comprises a heterogeneous cell population, and numerous factors are likely to be involved in disease outcome. Unfortunately, many patients with NMIBC are at high risk of disease recurrence and progression after primary treatment.[1, 2] The challenge for a clinician is to develop both reasonable surveillance protocols that provide cost-effective, noninvasive monitoring for low-risk patients and more aggressive approaches to identify high-risk refractory cancers before they progress.
While several molecular markers used to evaluate the development and prognosis of NMIBC have been studied, the limited value of these established markers has created the need for new molecular indicators of NMIBC. DNA hypermethylation-induced silencing of tumor suppressor and DNA repair genes is a frequent phenomenon in cancer, which has led to new opportunities for the understanding, detection, treatment, and prevention of the disease, including bladder cancer.[4-13] Although many of the different genetic or epigenetic changes that lead to aberrant gene expression in bladder cancer have been identified, the advent of high-throughput microarray technology makes it possible to gain a comprehensive insight into the molecular basis of human disease.[6, 14] Using this technology, genome-wide DNA methylation patterns and RNA expression levels in tumor specimens can be evaluated simultaneously, and tumor-cell specific molecular targets or gene classifiers can be identified.
NMIBC is increasingly regarded as a disease that cannot be treated solely on the basis of clinicopathological parameters. Identification of biomarkers that enable detection or predict the prognosis of NMIBC would be a valuable tool for guiding appropriate management strategies. Therefore, the aim of this study was to identify novel methylation markers that predict patient outcomes using microarray analysis of DNA methylation and RNA expression patterns in long-term follow-up NMIBC samples.
- Top of page
- Material and Methods
- Supporting Information
Analysis of aberrant DNA methylation is gaining traction in areas of cancer risk assessment, diagnosis, therapy monitoring, prognosis prediction, and novel drug targeting for several different types of cancer.[6-11] In this study, we used microarray analyses to detect novel epigenetic markers that are relevant to NMIBC and identified significant differences in the methylation patterns between NMIBC and NC. These candidate methylation markers showed close associations with unfavorable NMIBC features, including advanced stage and a higher grade. Notably, the methylation status of these candidate genes (either single or combined) was identified as an independent indicator for predicting prognosis.
Microarray-based clinical research has allowed comprehensive insights into the molecular basis of human disease, and new molecular targets at the whole-genome level are rapidly emerging. However, the identification of bona fide candidate methylation markers that have clinical relevance requires rigorous selection criteria, confirmation of the methylation status and gene expression levels in human tissues, and comparison with clinicopathological parameters or disease outcomes. For these reasons, DNA methylation status and gene expression levels were considered simultaneously when screening candidate genes in this study. The correlation between microarray methylation and PSQ analyses was checked, and concordant results were identified. Furthermore, methylation-induced gene silencing and prognostic outcomes were verified in long-term follow-up NMIBC patients. The results suggest that the novel methylation markers identified are specific to NMIBC and are appropriate for predicting prognosis.
There are an increasing number of reports showing that hypermethylation of individual genes can indicate different outcomes for bladder cancer patients.[9-12] However, a normal cell must undergo multiple genetic alterations before adopting a malignant and, ultimately, metastatic phenotype. Therefore, simultaneous assessment of multiple markers might better characterize the biological phenotype of a particular cancer. Additionally, the use of a single gene locus has several drawbacks. First, the maximum sensitivity can only be as high as the frequency of hypermethylation at a specific CpG locus. Second, noncancerous tissues can, in some cases, harbor CpG island hypermethylation at the same gene locus. Third, methylation of a single gene locus may occur in several cancers and, thus, be misleading. Furthermore, the number of hypermethylated genes increases as cancers progress and many methylation markers influence others[12, 13]; thus, cancer detection and prognosis are likely to be best estimated using a combination of markers.
The quantitative integration of methylation levels using a small set of hypermethylated genes is one of the major advantages of PSQ. Quantitative merging allows the quantitative comparison of samples and the accurate segregation of pathologic and prognostic covariates based on methylation levels. Merging the methylation levels of carefully selected genes yields maximum specificity and sensitivity. In this study, such quantitative combination enabled us to predict recurrence in a manner similar to that afforded by single markers; however, the power of multiple markers for predicting progression is much greater than that provided by single markers. These findings imply that the appropriate combination of selected genes will increase the usefulness of methylation-based biomarkers for both detection and prognosis.
A wide array of clinicopathological risk factors need to be considered when making a prognosis for NMIBC, including prior recurrence rates, tumor size, multiplicity, T-category, grade and treatment with intravesical therapy.[1, 2] In NMIBC, tumors with similar morphology may behave differently; thus, it is a major clinical challenge to accurately differentiate those patients whose tumor is likely to recur and/or progress after initial treatment from those in which recurrence and/or progression are unlikely. If candidate prognostic markers are to have considerable clinical relevance, they must provide a predictive capability beyond that offered by conventional clinicopathological parameters. Importantly, the methylation-based biomarkers identified in this study are independent predictors of prognosis, regardless of the clinicopathological characteristics of the NMIBC. However, considering modest value of area under curve and HR for the prediction of recurrence, although our results are promising, further validation study is necessary to reduce false predictive rates.
To the best of our knowledge, the current study is the first to identify HOXA9, ILS1 and ALDH1A3 as methylation-based prognostic markers of NMIBC. Although the relationships between these candidate markers and other types of cancer have been studied previously,[25-31] only one study looked at bladder cancer. HOXA9 and ILS1 are homeobox domain genes. HOXA9 plays an important role during development and hematopoiesis, and abnormalities are associated with leukemia, lung cancer, ovarian cancer, and bladder cancer.[13, 27-29] ILS1 affects various aspects of motor neuron identity, the lineage of cells of pancreatic endocrine origin, and cardiovascular progenitors.[32-34] ALDH1A3 is significantly associated with aldefluor positivity, the specific expression of which is a marker for breast cancer stem cells.[30, 31] A precise causal relationship between these putative genes (HOXA9, ILS1 and ALDH1A3) and NMIBC has not been yet determined. Additionally, even though these genes showed functional significance in bladder cancer, it does not always mean the crucial role in bladder tumor initiation or progression. Without clear demonstration of these kinds of association between these candidate markers and bladder cancer may be possible limitations of our study, but these will be elucidated with further study. Therefore, not only large-scale validation studies of human samples but also functional analysis and gene ontologic approach of these genes will give us more knowledge about their biological mechanisms and clinical relevance.
Because DNA methylation is reversible, it is thought to be a good therapeutic target. Drugs that target epigenetic alterations, such as DNA methylation inhibitors, restore the activity of genes by targeting aberrant heterochromatic regions, ultimately leading to the reactivation of tumor suppressor genes and/or other genes that are crucial for normal cellular function. The hypermethylation markers identified in this study showed significantly reduced expression levels in NMINC compared with NC, and methylation status was associated with prognosis. The identification of these methylation markers is noteworthy because of their potential utility for both detection and prognosis; they may also serve as candidate therapeutic targets.
From a clinical point of view, the most promising applications for epigenetic markers are clearly early detection, prediction of response to treatment and indication of disease prognosis. The results presented herein are promising because the candidate methylation markers were selected from a genome-wide analysis and validated in a relatively large number of human tissue samples obtained from long-term follow-up patients. In addition, the selected methylation markers are independent predictors of disease outcome. An accurate prediction of prognosis made using these candidate methylation markers would aid clinicians in terms of patient counseling, determining the frequency and extent of monitoring, and whether more aggressive therapy is needed. Further research will increase the accuracy of NMIBC detection and enable more realistic predictions regarding outcome. It may also lead to new therapies that target specific molecular defects, thereby significantly lowering the morbidity associated with NMIBC.
In conclusion, our findings suggest that the novel methylation markers HOXA9, ISL1 and ALDH1A3 are independent indicators of prognosis in NMIBC patients. These prognostic markers may constitute a promising tool for assessing the recurrence and progression of NMIBC patients and may facilitate the design of individualized therapeutic modalities.