G·A·B·B·A

Since prostate cancer is the most common malignancy in men in industrialized countries, and its incidence, morbidity and mortality have been increasing in recent years, this Thesis was aimed at identifying genetic abnormalities and define methodologies that may contribute for the early detection of prostate cancer.
In the first chapter of the Thesis, the potential of GSTP1 promoter methylation as a prostate cancer specific marker was investigated. Firstly, we hypothesized that quantitation of GSTP1 methylation by real-time methylation specific PCR (MSP) would allow for a distinction between neoplastic and non-neoplastic prostate tissue. Moreover, the feasibility of detecting this epigenetic alteration in voided urine was assessed in a preliminary study, and then conventional and real-time quantitative MSP analyses were performed to determine the clinical usefulness of GSTP1 hypermethylation quantitation as a prostate cancer specific marker in bodily fluids (voided urine and plasma). Finally, the relation between GSTP1 polymorphism and epigenetic alterations linked to GSTπ expression in prostate cancer was investigated.
In the second Chapter, analysis of promotor methylation in the endothelin B receptor gene (ENDRB) was performed in an attempt to increase the number of primary tumors amenable for screening.
The objective of the third Chapter of the Thesis was to investigate the frequency of mitochondrial mutations in prostatic adenocarcinoma (and paired PIN lesions), both in tissue samples and in bodily fluids, and whether these changes could be used as tumor molecular markers.

1. Quantitation of GSTP1 hypermethylation distinguishes between non-neoplastic prostatic tissue and organ confined prostate adenocarcinoma:
Tissue samples from 69 patients with early stage prostatic adenocarcinoma, 28 PIN lesions, and 31 patients with benign prostatic hyperplasia (BPH) were tested for GSTP1 hypermethylation by quantitative fluorogenic real-time MSP. To further verify the clinical applicability of this assay we performed a blinded investigation of prospectively collected prostate sextant biopsies of 21 patients with raised serum prostate-specific antigen (PSA) levels (11 with histologically identified adenocarcinoma, and 10 with no morphological evidence of adenocarcinoma). The median ratios (methylated GSTP1/MYOD1) found in resected hyperplastic prostatic tissue, intraepithelial neoplasia, and adenocarcinoma were 0.0, 1.4, and 250.8, respectively (P < 0.00001). The median GSTP1 methylation ratios found in adenocarcinomas and normal prostate tissue in sextant biopsies from the 21 prospective patients with high PSA levels also differed significantly (410.6 and 0.0, respectively; P = 0.0007). We concluded that quantitation of GSTP1 hypermethylation may augment standard pathology by accurately discriminating between normal hyperplastic tissue and prostatic carcinoma within a small tissue sample.

2. Detection of Prostate Cancer in Urine by GSTP1 Hypermethylation:
Matched specimens of primary tumor, peripheral blood lymphocytes (normal control) and a simple voided urine were collected from 28 patients with prostate cancer of a clinical stage amenable to cure. Genomic DNA was isolated from the samples and the methylation status of GSTP1 examined in a blinded manner using MSP. Decoding of the results revealed that 22 of 28 (79%) prostate tumors were positive for GSTP1 methylation. Remarkably, in 6 of 22 (27%) cases the corresponding urine sediment DNA was positive for GSTP1 methylation indicating the presence of neoplastic DNA in the urine. Furthermore, there was no case where a urine sediment DNA harbored methylation when the corresponding tumor was negative. Although we only detected GSTP1 methylation in under a third of voided urine samples, we have demonstrated for the first time that molecular diagnosis of prostate neoplasia in urine is feasible.

3. Quantitative GSTP1 hypermethylation in bodily fluids of prostate cancer patients:
Tissue samples from 69 patients with early stage prostatic adenocarcinoma and 31 patients with BPH were collected. Matched urine and plasma specimens were obtained preoperatively. After sodium-bisulfite treatment, extracted DNA was analyzed for GSTP1 promoter hypermethylation both by conventional and real-time quantitative MSP. In tissue samples, GSTP1 hypermethylation was detected in 63/69 (91.3%) of the cancer patients, and 9/31 of BPH patients (29%). Conventional MSP detected GSTP1 hyermethylation in a larger number of urine and plasma than real-time quantitative MSP (53.6% vs. 31.9%, overall). In all positive bodily fluids, the paired tumor was also confirmed to be methylated. GSTP1 hypermethylation was detected by both MSP methods in only 1 (3.2%) urine sample from a BPH patient. Although not quantitative, conventional MSP is currently more sensitive than real-time quantitative MSP in the detection of GSTP1 hypermethylation in bodily fluids from prostate cancer patients with clinically localized disease. The value of quantitative determinations in monitoring and risk assessment remains to be further explored.

4. I105V polymorphism and promoter hypermethylation of the GSTP1 gene in prostate adenocarcinoma:
To assess the risk of prostate cancer development, 3 populations comprising prostate cancer patients (PA), benign prostatic hyperplasia patients (PB) and healthy blood donors (PC) were enrolled and the respective GSTP1 genotype was determined. Tissue samples from the 105 PA patients (105 adenocarcinomas and 34 PIN lesions), and from 43 PB patients were tested for GSTP1 hypermethylation by conventional MSP. GSTπ expression was assessed by immunohistochemistry. No significant effect on prostate cancer risk was detectable for GSTP1 genotype, both comparing to the blood donors population (PC) (OR=1.13, 95% CI=0.62-2.06), and using the PB group as control population (OR=0.79, 95% CI=0.35-1.75). Moreover, no association was found between this genotype and tumor or BPH methylation status. In adenocarcinoma, a strong association between GSTP1 promotor hypermethylation and loss of GSTπ expression was observed. This trend was not retained in PIN or BPH lesions. We concluded that promoter hypermethylation is an effective cause of GSTP1 transcription silencing. Moreover, GSTP1 polymorphism is not associated with promoter hypermethylation nor with altered susceptibility to prostate cancer. It is suggested that epigenetic mechanisms may overcome the potential effects of GSTP1 variants in GSTπ activity.

5. Detection of endothelin receptor B hypermethylation in early stage prostate adenocarcinoma:
Prospectively collected tissue samples from 48 patients harboring clinically localized prostate cancer, and a control group of patients with BPH, were investigated. By MSP analysis, 83.3% of cases were methylated both in tumor and normal tissue of prostate cancer patients, as well as 91.3% of BPH samples. We concluded that EDNRB hypermethylation at CpG sites upstream the transcription start site does not distinguish normal from neoplastic prostate cells, thus precluding a role as prostate cancer marker.

6. Mitochondrial mutations in early stage prostate cancer and bodily fluids:
The D-loop region, 16S rRNA, and the NADH subunits of complex I were sequenced to identify mtDNA mutations in 16 matched PIN lesions and primary prostate cancers. Twenty mtDNA mutations were detected in the tumor tissue of three patients. Identical mutations were also identified in the PIN lesion from one patient. This patient with multiple point mutations also harbored a high frequency of microsatellite instability (MSI-H) in nuclear mononucleotide repeat markers. Remarkably, these mutations were also detected in all (3/3) matched urine and plasma samples obtained from these patients. Although mitochondrial mutations are less common in prostate adenocarcinoma, they occur early in cancer progression and they can be detected in bodily fluids of early stage disease patients.