Prostate cancer (PCa) remains a major medical health issue representing one of the frequent cancers among men. The discovery and widespread utilization of serum prostate-specific antigen (PSA) monitoring for early detection has greatly changed the way PCa is diagnosed and has resulted in the downstaging of the disease [1-5]. Active PCa screening with the use of serum PSA level monitoring and digital rectal examination is now widely prevalent in many countries [1,2]. The gold standard for PCa diagnosis is histopathologically reviewed prostatic biopsy cores. This technique, although generally effective, is subject to sampling errors and a significant false-negative rate [1,2]. Thus, the development of new tumor markers with better sensitivity and specificity to detect early cancer remains vitally important.

DNA hypermethylation-induced silencing of tumor suppressor and DNA repair genes is a frequent phenomenon affecting the hallmarks of cancer [6-9]. The genes undergoing methylation during the early phases of tumorigenesis could potentially be used as markers for identifying individuals at increased risk of developing malignancy or for aiding in the diagnosis of early malignancy, whereas those genes undergoing methylation during the progression of malignancy could potentially be used as prognostic markers [6-9]. These hypermethylation markers are promising tools for detecting cancer cells in tissue and body fluids, and various approaches for the detection of aberrant DNA methylation regions have been developed in recent years [7-10]. Most previous studies used methylation-specific polymerase chain reaction (MSP) or quantitative methylation-specific polymerase chain reaction (QMSP) to assess methylation status and often artificially categorized the data into simplistic hypermethylated or hypomethylated categories [7-9]. Despite common use, these methods have known disadvantages such as false-positive and false-negative results as well as the use of relative comparisons instead of absolute quantification [11,12]. To quantify DNA methylation, pyrosequencing (PSQ) is one of the most accurate techniques available [7,11,12].

CpG island methylation of glutathione-S-transferase-Pi (GSTP1) has been detected in several cancer types, including breast and hepatocellular cancer, but only in PCa is this abnormality consistently detected with a high specificity and rarely found in patients without disease [13-18]. The aim of the present study was to evaluate the relevance of GSTP1 methylation status in discriminating between benign prostatic hyperplasia (BPH) and PCa as well as its association with clinico-pathological parameters by use of a quantitative PSQ analysis.

Our results demonstrated that the methylation level of GSTP1 was significantly higher in PCa patients than in BPH patients. Moreover, GSTP1 hypermethylation was not only associated with an increased incidence of PCa, but was positively related to increases in the PSA level. In contrast, no significant relationships between GSTP1 methylation and age, stage, or Gleason score were established.

Methylation of a promoter region has been shown to be associated with suppression of gene transcription, and hence gene expression. CpG island methylation is thought to influence transcription by allowing the attachment of methyl-binding domain proteins, which indirectly block the binding of transcription factors [7-9,14]. Abnormal DNA methylation patterns are associated with many human tumor types, and differences in methylation patterns have also emerged as markers for cancer risk assessment, cancer diagnosis, and therapy monitoring in several different types of cancer [7-9]. Moreover, DNA methylation for cancer detection is quite powerful owing to the inherent stability of DNA compared with RNA or protein [8]. To date, aberrant methylation of CpG island-containing promoters of numerous hypermethylated loci including GSTP1 has been identified in PCa [7,10,14]. However, even in the same gene, the inconsistency of methylation levels reported in PCa could be the result of several factors, not the least of which is the different sample types used [7,13,14]. The variation could also be a result of different analytical methods and how they are applied, including the pretreatment of the DNA, the PCR conditions, and the PCR method. Recently, with technical developments, various methods have become available for the detection of the methylation status of certain genes in clinical samples [7,8]. Conventional MSP has been shown to have limited usefulness for specific cancer detection because benign lesions can be weakly positive and cannot be distinguished from cancer cases. Moreover, the results of MSP at a particular DNA region are simply reported as methylated or unmethylated, which may be also subjective, thus not allowing quantitation or identification of partial methylation. For improvement of sensitivity and quantitation, various techniques, such as nested two-stage PCR and QMSP, have been developed. However, these methods also have known drawbacks, such as false-positive and false-negative results, as well as the use of relative comparisons instead of absolute quantification [7,8,11,12]. In these regards, PSQ might be a best alternative tool, because it provides absolute quantitative information on bases at each interrogated CG site. Additionally, the assay design readily allows the interrogation of different parts of the gene(s) of interest as well as the inclusion of internal controls to address inaccuracies resulting from incomplete bisulfite conversion [7,11,12]. In the present study, we utilized PSQ to evaluate methylation status, and provided an optimal cutoff level to discriminate between PCa and normal controls. Our provided cutoff level was relatively low; thus, it might be misleading with the use of other techniques [7,11,12]. One of the major advantages of PSQ is the ability to quantitatively compare samples and to more accurately segregate varied pathologic covariates on the basis of methylation levels. If we did not use PSQ, we would not have found the significant correlation between GSTP1 methylation and PSA level.

GSTP1 is involved in the metabolism, detoxification, and elimination of potentially genotoxic foreign compounds, and thus acts to protect cells from DNA damage and cancer initiation. Suppression of GSTP1 activity can result in enhanced susceptibility to DNA damage and increased cancer incidence [13,14]. Many works have been reported on the development of GSTP1 methylation as a biomarker that could be used to detect PCa [13-18]. Most studies have reported a consistent link between GSTP1 methylation and PCa. Although the strength of the association has varied, in tissue samples taken by biopsy or during surgery, GSTP1 methylation has been detected in at least 70% of cases and is only rarely present in benign prostate tissue [13-18]. As previously mentioned, various reasons exist for this inconsistency in GSTP1 methylation levels, including the detection methods and different tissue types used. Our findings are consistent with these previous results and indicate that GSTP1 hypermethylation is a reliable indicator for the prediction of PCa.

Although histologically confirmed prostate tissues were used, the possibility of unrevealed PCa in BPH patients and a small fraction of undetected methylated DNA molecules might have affected our results for sensitivity and specificity. Nonetheless, in terms of methylation frequency, our results are promising. The sensitivity and specificity of GSTP1 methylation in distinguishing PCa and benign tissue reached 85.5% and 100%, respectively. Moreover, the frequency of GSTP1 methylation in PCa was independent of serum PSA level, Gleason score, and stage. Clearly, biopsy has no potential as a routine screening test because of its highly invasive nature. Although these findings are promising, multicenter, large-scale clinical validation studies with primary human cancer tissue and body fluids are currently underway at our institute to confirm GSTP1 as a diagnostic PCa methylation marker. These kinds of efforts will improve our understanding of the role of GSTP1 methylation in tumorigenesis and its clinical relevance.

The results of our study show that GSTP1 methylation is associated with the presence of PCa and PSA levels. This methylation marker represents a potentially useful indicator for the detection and monitoring of PCa.