A Case Study on Hypermethylation as a Prostate Cancer Indicator

Evaluation of APC Hypermethylation From Serum Samples as an Indicator for Presence of Prostate Cancer and Higher Gleason Score

Abstract

Purpose

In this prospective study it is aimed to identify role of hypermethlation analysis of APC gene from serum samples on detection of prostate cancer in patients with PSA level 2,5-10 ng/ml and its correlation with GS of the patients

Materials and Methods

Data from 142 patients underwent prostate biopsy with PSA level of 2,5-10 ng/ml was analyzed. Hypermethlation analysis of promoter region of APC gene from serum samples was done via methlation specific PCR. Patients were grouped as positive or negative depending on results of methylation analysis and compared for presence of prostate cancer and GS distrubution. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of methylation analysis were calculated. P value of 0.05 is used for statistical significance.

Results

There were 48 patients (33.8%) in methylation (+) group and 94 patients (66,2%) in methylation (-) group. Prostate cancer was detected in 43 of 142 (30,2%) patients after the first biopsy and 32 of these patients were in methylation (+) group. sensitivity and specificity of methylation analysis were 74,4% and 83,8%. PPV and NPV were 66.6% and 88.30 %. When GS distrubutions of the two groups analyzed patients in methylation (+) group were more likely to have higher GS compared to methylation (-) group(p=0,03).

Conclusions

Hypermethlation analysis APC gene from serum samples have shown promising results with its correlation with prostate cancer and higher GS. Further studies on prediciton of survival with long term follow up are needed to verify its value on prediction of survival

Introduction

Prostate Cancer (PCa) is one of the most important medical problems of the male population as it is the most common solid neoplasm in Europe having the highest incidence of 202,100 cases in 2004 (1). It is not only common but also a dangerous disease that it is the second most common cause of cancer death in men (2). These findings prove the importance of detection of PCa. However identifiying patients with clinically significant disease is an important issue, that is to treat patients that really need treatment while sparing ones that have silent disease.

The European Randomized Study of Screening for Prostate Cancer (ERSPC) trial concluded that 1,410 men would need to be screened and 48 additional cases of PCa would need to be treated to prevent one death from PCa (ERSPC-3). These findings point out that current diagnostic tools are inadequate for screening and detection of clinically significant PCa (3).

Epigenetic studies evolved in the era of urooncology in the last decade and DNA

methylation is widely studied for certain urological malignancies especialy for PCa (4). Hypermethylation of promoter regions of tumor supresor genes results in gene silencing. This epigenetic change in APC gene promoter regions has been shown in cancerous and precancerous prostate tissues,serum, and urine of PCa patients and sensitivity and specificity rates of 27-100% and 50-100% have been reported (5,6).

However the current literature lacks studies that investigate corrolation of results of methylation analysis from serum samples and clinicopathologic properties (grade and stage of disease) of patients. This point should be verified to identify applicability of this method in clinical practice.

In this prospective study it is aimed to identify role of hypermethlation analysis of promoter region of APC gene from serum samples on detection of prostate cancer in a population of patients with PSA level 2,5-10 ng/ml and its correlation with Gleason score (GS) of the patients.

Patients and Methods.

This study was approved by ethical committe of Turkish Ministry of Health. All patients signed informed consent. The patients admitted to urology outpatient clinics of Ankara University Schol of Medicine with PSA level of 2,5-10 ng/ml and who were decided to undergo prostate biopsy were involved in the study. Serum samples were obtained from all patients prior to biopsy and 12 cores transrectal ultrasound guided prostate biopsy was performed by a single radiologist.

Age, serum PSA level, prostate biopsy results were recorded and patients were followed regardless of the results of methylation analysis. Rebiopsy was suggested to patients with benign histology and persistant elevated PSA levels 6 months after the first biopsy. Patients with biopsy results of PCa were evaluted for staging and treated based on their stage. GS of the patients were evaluated in 3 groups GS:6 , GS: 7 and GS:8-10.

The patients were grouped as positive (methylation +) and negative (methylation -) based on methylation analysis results and compared with results of prostate biopsy. The primary outcome measures were sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of methylation analysis. As a secondary outcome measure the results were also compared with the GS groups.

Genetic analysis

DNA extraction Genomic DNA was extracted from peripheral blood samples using a PureLink ™ Genomic DNA Mini Kit (Invitrogen ™, catalog No. K1820-02) according to the manufacturer’s instructions and were stored at -20°C until modification and MSPCR amplification. DNA integrity and quantity was assessed with agarose gel electrophoresis and spectrophotometry, respectively. For this purpose, 1% agarose gel was used and 260 nm and 280 nm optical density ratio values (OD260/OD280) were determined.

Bisulfite Modification and Methylation-Specific PCR of APC and GSTPI promoters The genomic DNA was modified by sodium bisulfite treatment using the CpGenomeTM DNA Modification Kit (S7820, Chemicon, Temecula, CA). The DNA promoter methylation status of APC and GSTPI genes were investigated by MSPCR using the specific primers to methylated and unmethylated promoters. The methylated and unmethylated control DNA were also subjected to bisulfite DNA modification and PCR analysis, and serves as positive controls for methylated and unmethylated DNA respectively. H2O was used as negative control for the PCR reaction. The PCR reaction was started with denaturation at 95°C for 13 min, followed by 40 cycles at 95°C for 30 s, 56°C for 30 s, 72°C for 30 s; and 72°C for 5 min.

Statistical analysis

Continuous variables were compared using the t –test. Categorical variables were compared by the chi-square test..Sensitivity, specificity, PPV and NPV were calculated.

Results

Prostate biopsy was performed in 142 patients. The mean age of the patients were 68,3 ± 8,2 and mean ages of the methylated and unmethylated groups were not significantly different (67,8 vs 68,6 , p=0,47). There were 48 patients (33.8%) in group 1 and 94 patients (66,2%) in group 2. Prostate cancer was detected in 43 of 142 (30,2%) patients after the first biopsy and 32 of these patients were in methylation (+) group and 11 of them were in methylation (-) group. Based on these results, sensitivity and specificity of methylation analysis were 74,4% (95% CI: 58.83 % – 86.46 %) and 83,8% (95% CI: 75.08 % to 90.47 %). PPV and NPV were 66.6% (95% CI: 51.59 % to 79.59 %) and 88.30 % (95% CI: 80.02 % to 94.00 %).

When GS distrubutions of the two groups analyzed patients in methylation (+) group were more likely to have higher GS compared to methylation (-) group (p=0,041) and results are summarized in table 1. Second biopsy was performed in 12 (6 patients in each group) patients totally. At these second biopsies, PCa was observed in 4 patients, and all of them were in methylation (+) group.

Based on the total results of first and second biopsies, sensitivity and specificity ,

PPV and NPV were reevaluated. Sensitivity and specificity of methylation analysis were 76.6 % (95% CI: 61.97 % to 87.68 %) and 87.3 % (95% CI: 78.97 % to 93.29 %). PPV and NPV were 75.0 % (95% CI: 60.4 % to 86.3 %) and 88.30 % (95% CI: 80.02 % to 94.00 %).

Discussion

Epigenetic alterations, such as hypermethylation of prompter regions of tumor supressor genes are associated with many human tumor types and this type of alteration have also emerged as markers for cancer risk assessment, cancer diagnosis, and therapy monitoring in several different types of cancer (7-10). APC is a well-characterized tumor suppressor gene and it downregulates Wnt signaling by targeting the transcriptional coactivator beta-catenin for proteasomal degradation and this prevents its association with the nuclear transcription factor Tcell factor/lymphoid enhancer factor (11). The activation of Wnt pathway leads to deregulation of cell proliferation and differentiation and is a common feature of many human cancers including prostate cancer (12-14).

Detection of clinically significant PCa at an early stage is still a great challenge and current imaging modalities are often not able to detect a tumor and biopsies may miss the tumor focus. Role of gene promoter hypermethylation in prostate carcinogenesis, has been studied recently and promising results to improve PCa diagnosis has been established (5-7).

In this study methylation analysis of promoter region of APC gene is performed prior to prostate biopsy in serum samples in order to evaluate the the role of this analysis on prostate cancer detection. Serum samples were obtained from all patients prior to biopsy and PCa was detected in siginificantly higher rates in patients with positive methylation analysis. In their study Yoon et al. showed that APC methylation levels were significantly higher in PCa patients than in benign prostate hyperplasia patients. Moreover, APC hypermethylation was not only associated with the increased incidence of PCa but was also positively correlated with increased GS (15). This has also been shown in other studies indicating APC hypermethylation as a reliable predictor of PCa and of its aggressive feature (12,13). Similarly, in our study, methylation positive patients were found to have higher GS and GS>7 was not observed in any patient with negative methylation analysis.

Sensitivity and specificity of APC hypermethylation has been studied on prostate tissue samples and high sensitivity (89.3%) and specificity (98.1%) rates have been reported (15). In our study, sensitivity of 74,4% and specificity of 83,8% was found. These results are slightly lower compared to results of Yoon et al. and this may be due to source of DNA, that is higher rates might be reached when analysis is made through prostate tissue samples compared to serum samples. Additionally the results may be effected from the detection method, including the primer design, reagents, detectors, equipment, and protocols, all of which potentially influence the sensitivity and specificity.

The important point of our study is making the analysis from serum samples to evaluate its role in clinical practice. The lack of such studies has been emphasized in previous trials conducted on tissue samples (15). Repat biopsies is an important issue in PCa diagnosis in the study of Trock et al. (16) APC methylation provided a very high NPV with a low percentage of false negatives, in a clinical cohort of men undergoing repeat biopsy.In our study, repeat biopsy was performed in 12 patients (6 patients in each group) and all of the 4 patients with PCa detected in second biopsy were in the methylation positive group. Our study involves low number of repeat biopsies therefore further studies with higher number of patients evaluating role of hypermethylation analysis from body fluids should be conducted on the topic of repeat biopsies.

Conclusion

Prediction of clinical significance of PCa is of clinicial importance and hypermethlation analysis of promoter region of APC gene from serum samples have shown promising results with its correlation with prostate cancer and higher GS. Further studies on prediciton of survival with long term follow up are needed to verify its value on prediction of survival.

Conflict of interest: None of the authors have any conflict of interest

Ethical standarts: The study was approwed by ethical committe of Turkish Ministry of Health.

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Table 1. GS distrubutions of the two groups