Abstract
Aim: The aim of this study was to evaluate the diagnostic potential of the quantification of glutathione-S-transferase P1 (GSTP1) gene hypermethylation in molecular detection of prostate cancer in tissue biopsies. Methods: One hundred fourteen male patients were enrolled in the study; 44 patients with histopathologically confirmed prostate adenocarcinoma, 20 patients with variable degrees of prostate intraepithelial neoplasia, and 50 with benign prostatic hyperplasia, who served as a control group. Real-time quantitative methylation-specific polymerase chain reaction was used for assessment of methylation of the promoter region of the GSTP1 gene. Results: Methylation of the GSTP1 promotor was detected in 24% of patients with benign prostatic hyperplasia, 60% of patients with prostate intraepithelial neoplasia, and in 86.3% of prostate adenocarcinoma patients. A statistically significant difference in the GSTP1/MYOD1 (myogenic differentiation 1gene) methylation ratios among the three groups was observed (p=0.0001). At the cutoff value of 9, GSTP1/MYOD1 methylation ratios showed sensitivity in the detection of prostate adenocarcinoma of 71.8% and specificity of 96%. Conclusions: Methylation of the GSTP1 promotor is a common molecular alteration in prostate cancer that may be a useful adjunct to serum screening tests and digital rectal examination findings and the use of quantitative real-time methylation-specific polymerase chain reaction is a promising technique that often distinguishes malignant from nonmalignant prostate disease.
Introduction
P
Although serum concentration of prostate-specific antigen (PSA) remains important for disease diagnosis while still organ-confined, in addition to its use for monitoring recurrence or progression after definitive therapy (Beatty, 2004), the utility of PSA and digital rectal examination (DRE) for identification of patients at risk for harboring PCa is limited by low specificity as the test is unable to discriminate between carcinoma and benign lesions resulting in a high frequency of unnecessary biopsies; thus, the detection of low-volume PCa remains a diagnostic challenge (Bock and Klee, 2004; Thompson et al., 2004; Pfister and Basuyau, 2005). Highly specific, minimally invasive techniques that are cost effective for screening and monitoring would facilitate earlier diagnosis of PCa and may also reduce the number of unnecessary biopsies (Catalona et al., 2000).
As the progression of PCa and other malignancies is characterized by increased genetic and epigenetic aberrations that are not usually found in normal DNA (Sunami et al., 2009), the use of epigenetics, specifically DNA methylation as a cancer biomarker, offers several advantages. Methylation of regulatory sequences at the glutathione-S-transferase P1 (GSTP1) gene locus is found in the vast majority of prostate carcinomas and it may be identified even in preneoplastic lesions (Esteller et al., 2001; Lin et al., 2001; Jerónimo et al., 2002; Jones and Baylin, 2002).
Methylation of CpG islands, which are GC-rich regions of DNA located within the promoter region of the GSTP1 gene, may result in its epigenetic silencing, and therefore downregulation of transcriptional activity, and as GSTs comprise a family of enzymes involved in DNA protection from metabolites of carcinogens and reactive oxygen species by conjugating them to glutathione, detoxification and elimination of potentially genotoxic foreign compounds is suppressed by CpG island methylation, resulting in enhanced susceptibility to DNA damage and increased cancer incidence (Lin et al., 2001; Henrique and Jerónimo, 2004; Phé et al., 2010; Jerónimo et al., 2001).
Materials and Methods
The study approval was obtained from the ethics committee of the Faculty of Medicine, Alexandria University. All patients provided signed, written informed consent to participate in this study. Serum PSA level was measured using the Advia™ Centaur Immunoassay System.
Specimen collection and DNA isolation
A total of 114 male patients were enrolled in the study: 44 patients with histopathologically confirmed prostate adenocarcinoma, 20 patients with variable degrees of prostate intraepithelial neoplasia (PIN) but without invasive carcinoma, and 50 with benign prostatic hyperplasia (BPH), who served as a control group.
BPH biopsies were obtained through trans-urethral resection of the prostate procedures. Pathological classification of patients with localized prostate adenocarcinoma was done according to the TNM staging system (American Joint Committee on Cancer (2002) Prostrate: in AJCC Cancer Staging Manual) and Gleason microscopic grading system. The two-grade system was used to classify biopsies of PIN samples: low-grade PIN (corresponding to grades I and II) and high-grade PIN (corresponding to grade III). Of the 20 PIN biopsies, 6 only were low-grade PIN and the rest of the samples (n=14) were high-grade PIN.
Genomic DNA was isolated from snap-frozen tissue biopsies using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA).
Bisulfite treatment
Genomic DNA was treated with sodium bisulfite using the Imprint DNA Modification Kit (Sigma-Aldrich, St. Louis, MO). DNA was denatured at 99°C for 6 min followed by 90 min at 65°C. The sodium bisulfite treatment of DNA converts unmethylated cytosines to uracils, whereas methylated cytosines remain unchanged. Bisulfite treatment was followed by clean-up of modified DNA according to the manufacturer's instructions and the modified DNA was stored at −20°C until further processing.
Real-time quantitative methylation specific polymerase chain reaction
Sodium bisulfite-treated genomic DNA was amplified by quantitative fluorescence-based real-time methylation specific polymerase chain reaction (Q-MSP) using TaqMan technology as described previously (Jerónimo et al., 2002).The sequences of the primers and probe used to amplify and detect methylated GSTP1 and MYOD1 (myogenic differentiation 1) (internal reference gene) are listed in Tables 1 and 2.
Oligonucleotide primers amplify bisulfite-converted DNA within the 3′-end of the promoter of the GSTP1 gene (gene of interest), whereas for the internal reference gene, MYOD1, the primers and probe amplify and detect a region of the gene that is devoid of CpG nucleotides. Thus, amplification of MYOD1 by QMSP occurs independent of its methylation status, whereas the amplification of GSTP1 is proportional to the degree of cytosine methylation within the GSTP1 promoter. Methylation ratio was used as a measure of the relative level of methylated GSTP1 DNA in each sample and was estimated as the ratio of the fluorescence emission intensity values for the GSTP1 PCR products to those of the MYOD1 PCR products obtained by TaqMan analysis, multiplied by 1000 for easier tabulation. A separate amplification assay was performed for each of GSTP1 and MYOD1. The final reaction volume was 25 μL containing the sense and antisense primers at 600 nM each, the probe at 200 nM, 1× TaqMan universal PCR master mix No AmpErase UNG containing AmpliTaq Gold DNA polymerase, and 3 μL bisulfite-converted genomic DNA. PCR cycling conditions were: 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Fluorogenic quantitative real-time MSP assays were performed on the Mx3000P™ Real-Time PCR System (Stratagene, CA). A standard curve was created using serial dilutions of the positive control DNA (EpiTect Control DNA-human; QIAGEN) that contains 10 ng/μl methylated and bisulfite converted human DNA.
To ensure the specificity of the MSP analysis, a negative control (No template control) and a positive control (using EpiTect Control DNA) were included in each run.
Statistical analysis
Statistical analysis was performed using MedCalc for Windows, version 11.3.3.0 (MedCalc Software, Mariakerke, Belgium). Nonparametric tests were used: GSTP1 methylation levels in the studied groups were examined using Kruskal-Wallis test. Spearman's rho correlation test was used to assess the relation between continuous variables and χ2 test was used for categorical variables. The cutoff value for methylation threshold of the GSTP1 gene was established by receiver operating characteristic analysis. Sensitivity, specificity, and area under the curve were calculated. Statistical significance was set at p-value < 0.05.
Results
Pathological criteria of adenocarcinoma patients are illustrated in Table 3. Post-hoc analysis showed that the methylation level in adenocarcinoma biopsies was significantly higher than in biopsies from PIN and from BPH and also that methylation ratio was higher in PIN than BPH patients (p<0.05) (Table 4). Using receiver operating characteristic curve analysis, a cutoff level of 9 was set for GSTP1/MYOD1 ratios to distinguish benign from malignant tissue because it represents an optimal balance between the sensitivity and the specificity of the test. At a cutoff value of 9, the sensitivity of MSP quantitation of GSTP1 methylation in the detection of prostate adenocarcinoma was 71.8% (95% CI: 53.3-86.3), specificity 96% (95% CI: 79.6-99.9), positive predictive value 95.8%, and negative predictive value 72.7%.
Data are presented as number (%).
Data are presented as median (min-max) and were analyzed by Mann-Whitney test, p<0.05 is statistically significant.
Data are presented as number (%).
BPH, benign prostatic hyperplasia; PIN, prostate intraepithelial neoplasia; PSA, prostate-specific antigen.
A significant association was found between GSTP1 methylation status and the pathologic stage (p=0.014) and the Gleason score (p=0.011) for adenocarcinoma patients.
Discussion
Methylation of the CpG island region of the GSTP1 gene is associated with suppression of its activity resulting in enhanced susceptibility to DNA damage and increased cancer incidence in >90% of all PCa cases (Song et al., 2002; Dobosy, 2007).
In the present study, Q-MSP was used to assess GSTP1 promoter hypermethylation in 44 adenocarcinoma tissue biopsies, 20 PIN biopsies, and 50 BPH biopsies. Most adenocarcinoma patients were of relatively advanced stages. This can be attributed to the limited value of the available techniques in early disease detection. Serum PSA has limited diagnostic value because the sensitivity and specificity of the test is at best 75% and in addition it is influenced by age-related benign prostatic hypertrophy. The value of DRE and imaging techniques are also limited in early disease detection, due to restrictions inherent to physical examination of the prostate gland and the lack of distinctive radiologic or ultrasonographic features (Fradet, 2009). Recent refinements of molecular biology techniques have led to the development of a series of new tests that, hopefully, will improve diagnostic accuracy beyond serum PSA, DRE, and radiological diagnosis aiming at earlier disease detection (Fradet, 2009). Epigenetic alterations are considered an emerging class of cancer biomarkers as most of the common human tumors appears to have one or more hypermethylated loci (Henrique and Jeronimo, 2004).
In an attempt to reach the optimum in early gene methylation detection, sample type and detection techniques are modified as well as the recently targeted approach of profiling multiple genes silenced by hypermethylation as a panel (Bastian et al., 2007)
Multiple types of clinical samples have been tested in variable gene methylation assessments, including serum/plasma, semen ejaculates, urine voided postprostatic massage, or postprostatic biopsy and prostatic tissue samples (Jerónimo et al., 2002). Despite the fact that urine and plasma are good matrices for screening methylation assays, using either plasma or urine was always hindered by the low DNA content of both impairing methylation detection. Jerónimo et al. (2002) reported a low methylation frequency of 18.8% for urine sediments and 13% of plasma DNA samples from patients with PCa using Q-MSP, and even using conventional-MSP (C-MSP), they were able to detect GSTP1 methylation in only 30.4% and 36.2% urine and plasma samples, respectively.
In a trial to increase DNA yield, some researchers as Goessl et al. (2001) collected urine samples after prostatic massage and were able to detect GSTP1 promotor hypermethylation in 78% of patients with prostate adenocarcinoma. However, Gonzalgo et al. (2003) detected GSTP1 hypermethylation in 39% only of postprostatic biopsy urine specimens of PCa patients.
Prostate tissue biopsy was able to detect GSTP1 methylation in 70.5% to 94% of PCa patients (Goessl et al., 2000; Jerónimo et al., 2002; Steiner et al., 2010), with the variation between studies mostly due to variation in DNA methylation detection techniques.
In agreement with these studies, the present study, based on Q-MSP and performed on prostatic biopsies, detected methylation in 24% of patients with BPH, 60% of patients with PIN, and in a high percentage of prostate adenocarcinoma patients (86.3%).
As molecular detection of gene methylation needs highly refined and accurate techniques, the frequently used ones are C-MSP and Q-MSP with multiple advantages for each. In a study by Jerónimo et al., Q-MSP was used to quantify the GSTP1 methylation level in voided urine and plasma of two groups of patients: one with clinically localized cancer and a control group of patients with BPH. Then, the results were compared with those of C-MSP. They reported a larger number of samples positive for GSTP1 hypermethylation using C-MSP compared to Q-MSP (53.6% vs. 31.9%) and suggested that the former method is more sensitive than the latter, perhaps because of the greater specificity of the probe designed for quantitative analysis and the high back-ground fluorescence intrinsic to Q-MSP analysis (Jerónimo et al., 2002).
The limitations of using C-MSP for the detection of GSTP1 promoter methylation to detect cancer are mainly due to the positive GSTP1 methylation score of many benign prostatic hyperplastic lesions as well as actual cases of PCa (Jerónimo et al., 2002).
Using Q-MSP, the present study demonstrated a clear difference in GSTP1 methylation levels between benign and neoplastic prostate tissues at a cutoff value set at a methylation level of 9 with a sensitivity of 71.8% and a specificity of 96%.
In agreement with these findings, Harden et al. (2003) used Q-MSP technique in comparing the use of histology alone versus histology and methylation assays in detection of cancer in 72 sextant biopsy samples. They reported an increase of 11% in sensitivity of detection of malignancy (75% sensitivity) with no change in specificity (100% specificity) upon using combined histology and GSTP1 Q-MSP at a methylation assay threshold >10 and on decreasing the cutoff to 5, sensitivity improved to 79%; a 15% improvement over histology alone.
Limitations of the present study include the relatively small patient population and the lack of long-term follow-up. Repeat biopsy results of patients in our cohort with a diagnosis of no cancer, atypia, or PIN are needed to validate the utility of GSTP1 methylation for detection of early PCa. Using of a panel of methylated genes will mostly allow earlier and better identification of PCa.
Conclusion
Methylation of the GSTP1 promoter is a common molecular alteration in PCa that may be a useful adjunct to serum screening tests and DRE findings, and the use of quantitative real-time methylation-specific PCR is a promising technique that often distinguishes malignant from nonmalignant prostate disease.
Footnotes
Disclosure Statement
No conflicts of interest to be reported.