European Alliance On Prostate Cancer

Anatomy Prostatic Cancer
Fig. 4. Random sampling core needle biopsies of the prostate peripheral zone region can miss prostatic carcinomas. In a gland with multifocal prostate cancer, biopsy #1 detects a carcinoma lesion whereas biopsy #2 misses two carcinomas.

permits prognostic assessment of prostate cancer natural history, the use of minute core needle biopsy tissue fragments presents considerable challenges to diagnostic surgical pathologists (35). For these reasons, new molecular biomarkers, capable of increasing the sensitivity and specificity of prostate cancer screening, improving the accuracy of prostate cancer diagnosis, and providing stratification for treatment recommendations, are needed.

Can somatic changes in DNA methylation patterns serve as useful prostate cancer molecular biomarkers? Clearly, with PCR technologies, nucleic acid markers are detectable with extraordinary sensitivity, often in the range of a single molecule, and, of the nucleic acids, DNA may be superior to RNA in terms of stability through specimen collection and sample handling. Thus, both genetic and epigenetic genome alterations have been pursued as molecular biomarkers for all human cancers (51,52). DNA methylation changes are particularly attractive for this purpose for most human cancers, because unlike point mutations, for example, CpG island hypermethylation changes seem more consistent from case to case, permitting a single assay to be used to detect all cases (2). Prostate cancer is no exception; there have not been any common point mutations in any genes yet described, however, several consistent CpG island hypermethylation changes have been reported (40,41).

Currently, two major strategies for the detection of CpG dinucleotide methylation changes are most commonly used, although new strategies are under active development. The first detection method features the use of restriction endonucleases that cut recognition sites differently if the sites contain 5-meCpG. This method has been used along with Southern blot analysis and with PCR to discriminate DNA methylation changes at particular genome sites (53). The second detection approach uses sodium bisulfite modification to facilitate the selective deamination of C, but not of 5-meC, to U, creating a DNA sequence difference at 5-meC vs C after PCR amplification. This approach has been used for mapping and sequencing of 5-meC at particular genome sites, and along with PCR, featuring primers specific for bisulfite-converted 5-meC-containing or C-containing sequences (54,55). Each of these approaches has strengths and weaknesses as biomarker detection assays. Assays using 5-meCpG-sensitive restriction enzymes and PCR are spectacularly sensitive, capable of detecting single hypermethylated CpG island sequences, but are prone to false-positive results, attributable to inefficient cutting of unmethylated sequences. In contrast, bisulfite modification and PCR (MS-PCR) is somewhat less sensitive, because the bisulfite modification procedure can damage DNA sequence targets, but is often more specific (40).

For prostate cancer, the greatest amount of attention has been afforded to GSTP1 CpG island hypermethylation, the first alteration reported and a change present in greater than 90% of cases that have been carefully evaluated (28,31,53). The somatic genome change has proven remarkably robust as a candidate molecular biomarker. Using a variety of different detection strategies, in some 24 studies with 1071 cases, GSTP1 CpG island hypermethylation has been detected in DNA from prostate tissues in more than 81% of cases analyzed, with the sensitivity of detection varying somewhat, depending on assay (40). GSTP1 CpG island methylation changes are conspicuously absent from all normal human tissues, including normal prostatic epithelial cells isolated by laser capture microdissection, but are commonly present in prostate cancer precursor lesions, such as PIA and prostatic intraepithelial neoplasia (33,36,37). Thus, assays capable of detecting GSTP1 CpG island hypermethylation in DNA from prostate tissues have the potential of discriminating prostate cancer and its precursors from other prostate abnormalities, and of detecting neoplastic cells even when not readily evident by microscopy (56). Furthermore, such assays have been found to detect prostate cancer DNA in prostatic secretions, permitting the use of urine specimens for prostate detection and diagnosis (57-59).

In addition to the GSTP1 CpG island, CpG island sequences at more than 40 other gene sites have been evaluated for hypermethylation changes in prostate cancer DNA (Table 1). By examining many CpG islands for each prostate cancer case, CpG island hypermethylation "profiles," distinct from other cancers, have emerged (40,41). Using the quantitative MS-PCR technology, the detection of CpG island hypermethylation at combinations of sites, including GSTP1, APC, RASSF1a, PTGS2, and MDR1, has been reported to distinguish prostate cancer from noncancerous tissue with sensitivities of 97.3 to 100% and specificities of 92 to 100% (41). It is likely that more somatic targets of CpG island hypermethylation will be discovered in the future; many such genes may offer new opportunities for molecular biomarkers that can be used in prostate cancer detection and diagnosis. Will some sort of DNA methylation assay become a prostate cancer screening tool? The best opportunity seems to be sensitive detection of somatic DNA methylation changes in urine, if the urine can be collected in such a way that it contains prostate secretions (57).

In addition to aiding in prostate cancer detection and diagnosis, somatic DNA methylation changes may provide prognostic information. Both similarities and differences have been reported for patterns of DNA methylation in primary vs metastatic cancers (41). Perhaps the somatic DNA methyla-tion changes characteristic of metastatic prostate cancers can be detected in a fraction of primary prostate cancers at high risk for metastatic progression. Thus far, hypermethylation of CpG island sequences at EDNRB, RARp, RASSF1a, ERp, and T1G1 have been correlated with primary prostate cancer stage and/or grade, known prognostic markers (41,60-63). Furthermore, differences in PTGS2 CpG island hypermethylation in DNA from primary prostate cancers have been found to independently predict prostate cancer recurrence after radical prostatectomy, even when tumor grade and stage were considered (41) (Fig. 5). In a conceptually different approach, sensitive detection of somatic changes in DNA methylation may add great sensitivity to conventional prostate cancer staging. As described above, GSTP1 CpG island hypermethylation changes are so common in prostate cancer cells, and so rare elsewhere, that detection of DNA with such changes in blood, lymph nodes, bone marrow, and so on, from a man known to have prostate cancer likely indicates the presence of cancer at these sites. In an example of this strategy, serum DNA showing GSTP1 CpG island hypermethyla-tion is more readily detected in men with known metastatic prostate cancer than without. Furthermore, for men with localized prostate cancer, the presence of prostate cancer DNA, identified by assays of GSTP1 CpG island hypermethylation, portends prostate cancer recurrence after radical prostatectomy (64).

Ultimately, the use of detection strategies for somatic DNA methylation changes can be imagined for prostate cancer screening, detection, diagnosis, prognosis, and treatment stratification. To best exploit the opportunity presented, a more complete knowledge of DNA methylation changes in prostate cancer cases, and the correlations of such changes with neoplastic transformation, with prostate

Fig. 5. PTGS2 CpG island hypermethylation as a prognostic molecular biomarker for recurrence risk after radical prostatectomy. Quantitative MS-PCR detection of PTGS2 CpG island hypermethylation stratifies men into low (below the median) and high (above the median) risk for prostate cancer recurrence after radical prostatectomy (p = 0.0017). Multivariate analyses indicated that quantitative PTGS2 CpG island hypermethylation predicted prostate cancer recurrence independently of tumor stage and grade (41).

Fig. 5. PTGS2 CpG island hypermethylation as a prognostic molecular biomarker for recurrence risk after radical prostatectomy. Quantitative MS-PCR detection of PTGS2 CpG island hypermethylation stratifies men into low (below the median) and high (above the median) risk for prostate cancer recurrence after radical prostatectomy (p = 0.0017). Multivariate analyses indicated that quantitative PTGS2 CpG island hypermethylation predicted prostate cancer recurrence independently of tumor stage and grade (41).

cancer grade and stage, with malignant progression and metastasis, and with response to treatment, will need to be determined. In addition, assay strategies for detecting such changes will need to be refined. Finally, new approaches to biospecimen collection and handling, such as the recovery of prostate secretions, may also prove helpful.

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