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Molecular Cancer Research 4:1-8 (2006)
© 2006 American Association for Cancer Research

Cancer Genes and Genomics

Epigenetic Heterogeneity of High-Grade Prostatic Intraepithelial Neoplasia: Clues for Clonal Progression in Prostate Carcinogenesis

Rui Henrique1,2,5, Carmen Jerónimo1,2,3,6, Manuel R. Teixeira3,5, Mohammad O. Hoque1, André L. Carvalho1, Irene Pais2, Franclim R. Ribeiro3, Jorge Oliveira4, Carlos Lopes5 and David Sidransky1

1 Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins University School of Medicine, Baltimore, Maryland and Departments of 2 Pathology, 3 Genetics, and 4 Urology, Portuguese Oncology Institute-Porto; 5 Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto; and 6 Fernando Pessoa University School of Health Sciences, Porto, Portugal

Requests for reprints: Carmen Jeronimo, Department of Genetics, Portuguese Oncology Institute-Porto, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal. Phone: 351-225084000; Fax: 351-225084016. E-mail: cjeroni{at}ufp.pt


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Patients and Methods
 References
 
High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate adenocarcinoma, but the frequency and timing of epigenetic changes found in prostate carcinogenesis has not been extensively documented. Thus, the promoters of three genes (APC, GSTP1, and RARß2) involved in prostate carcinogenesis were tested by quantitative methylation-specific PCR in tissue DNA from 30 prostate carcinomas, 128 high-grade PIN lesions, and 30 normal prostate tissue samples dissected from 30 radical prostatectomy specimens using laser capture microdissection. The percentage of methylated alleles (PMA) was calculated for each gene, and hierarchical cluster analysis was used to define the degree of similarity of epigenetic alterations among the various samples. We found that PMA values of APC and RARß2 were higher than those of GSTP1 in all three types of tissue samples and median PMA values for all three genes were higher in prostate cancer. By cluster analysis, 26 of 30 prostate carcinomas and 82 of 128 high-grade PIN lesions were grouped in the "high methylation" branch, whereas 24 of 30 normal prostate tissue samples were allocated in the "low methylation" branch. Although high-grade PIN lesions are epigenetically more similar to prostate carcinoma than to normal prostate tissue, paired prostate carcinoma and high-grade PIN lesions did not always segregate together. We concluded that APC and RARß2 hypermethylation is frequent in normal prostate tissue and the progressive enrichment in cells carrying methylated alleles observed in high-grade PIN and prostate carcinoma is consistent with clonal progression. Because GSTP1 promoter methylation is mainly observed in prostate carcinoma and some high-grade PIN lesions, it represents an important marker for the transition of in situ to invasive neoplasia. (Mol Cancer Res 2006;4(1):1–8)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Patients and Methods
 References
 
Prostate cancer is a leading cause of cancer-related mortality and morbidity in western countries (1). Despite major research efforts, the genetic mechanisms underlying early prostate cancer development and progression are still largely unexplored. Because the clinical behavior of this neoplasm is highly variable, a better understanding of the molecular alterations associated with prostate carcinogenesis is expected to contribute to improved diagnosis, clinical management, and outcome prediction.

Over the years, several putative prostate cancer precursor lesions or conditions have been identified, including prostatic intraepithelial neoplasia (PIN), atypical adenomatous hyperplasia, and proliferative inflammatory atrophy (2). Among these conditions, high-grade PIN stands as the most likely and well-documented prostatic precancerous lesion (2), preceding at times by several decades the development of invasive prostate cancer (3). Indeed, several studies suggested that high-grade PIN is more closely related to prostate carcinoma than normal prostate tissue (4, 5). Moreover, high-grade PIN lesions, like prostate carcinomas, display considerable genetic heterogeneity as revealed by loss of heterozygozity, fluorescence in situ hybridization, gene expression profiling, and comparative genomic hybridization studies (6-13).

Recently, hypermethylation of CpG islands within the promoter and/or 5' regions of genes was recognized as a common alteration in cancer-related genes often associated with partial or complete transcriptional disruption (14). This epigenetic alteration provides an alternative pathway to gene silencing in addition to gene mutation or deletion. We and others reported that promoter methylation of several genes is a common feature of prostate cancer and high-grade PIN (15–20) and a new molecular marker for early cancer detection (21). Likewise, previous studies suggested that high-grade PIN lesions are epigenetically heterogeneous and that differential methylation of cancer-related genes might occur during histopathologic progression to prostate cancer (22).

We sought to characterize the frequency and pattern of epigenetic changes in the progression from normal prostate epithelium through high-grade PIN and prostate cancer. We thus tested three target gene promoters (APC, GSTP1, and RARß2) reported previously to be differentially hypermethylated in benign, preneoplastic, and cancerous prostate tissues (19, 20). For that purpose, matched samples of prostate carcinoma, multiple high-grade PIN lesions, and morphologically normal prostate tissue were obtained from 30 prostatectomy specimens. Laser capture microdissection was used to obtain a highly purified sample of target epithelial cells from each lesion, and quantitative methylation-specific PCR (MSP) allowed us to begin to order these key epigenetic changes in the progression to high-grade PIN and prostate cancer.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Patients and Methods
 References
 
Clinical and Pathologic Data
We obtained tissue samples from 30 patients with clinically localized prostate adenocarcinoma, with a median age of 65 years (range, 50-72). The median value of the preoperative serum PSA was 7.8 ng/mL (range, 3.01-14.5) and the median Gleason score of the prostate adenocarcinomas was 6 (range, 5-7). Three (10%) cases were staged as pT2a, 17 (56.7%) cases as pT2b, 9 (30%) cases as pT3a, and 1 (3.3%) case as pT3b. Overall, 128 distinct high-grade PIN lesions were identified and microdissected by laser capture in the 30 radical prostatectomy specimens [range, 1-12 lesions in individual cases (median, 4)].

Quantitative MSP in Prostatic Tissues
The frequency of methylation and median (interquartile range) percentage of methylated allele (PMA) values for each gene in prostate carcinomas, high-grade PIN lesions, and normal prostate tissue samples is listed in Table 1 and graphically displayed in Fig. 1 .


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  		Table 1. Frequency of Positive Cases and Distribution of the PMA [Median (Interquartile Range)] in Prostate Carcinoma, High-Grade PIN, and Normal Prostate Tissue

 

Figure 1
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  		FIGURE 1. Distribution of the PMA for (A) APC, (B) GSTP1, and (C) RARß2 in normal prostate tissue (NPT; n = 30), high-grade PIN (HGPIN; n = 128), and prostate carcinoma (PCa; n = 30).

 
All prostatectomy specimens carried at least one high-grade PIN lesion methylated at the RARß2 promoter, whereas 29 (96.7%) and 18 (60%) surgical specimens carried at least one high-grade PIN lesion methylated at the APC and GSTP1 loci, respectively.

Statistically significant differences in PMA among the three groups of tissue samples were noted for all the target genes (GSTP1, APC, and RARß2; P < 0.0001 in all cases). Generally, prostate carcinoma displayed the highest median PMA and these statistically differed from those of high-grade PIN and normal prostate tissue for APC, GSTP1, and RARß2 (P < 0.0001). Except for GSTP1, the PMA of high-grade PIN and normal prostate tissue also differed (P < 0.001 for APC and P < 0.00001 for RARß2). As a rule, the APC and RARß2 PMA was higher than GSTP1 PMA within an individual tissue. An example of the distribution of methylation at the three target genes in multiple lesions from a single prostate is schematically depicted in Fig. 2 .


Figure 2
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  		FIGURE 2. Schematic representation of radical prostatectomy specimen RP17 showing a prostate carcinoma, six lesions of high-grade PIN, and normal prostate tissue from the peripheral zone (normal prostate tissue). The PMA of each of the three target genes (APC, GSTP1, and RARß2) is depicted for each tissue sample. Note that only a high-grade PIN lesion shows the same epigenetic profile of the prostate carcinoma.

 
Hierarchical Cluster Analysis
The results from hierarchical cluster analysis of quantitative MSP results for 30 prostate carcinomas, 128 high-grade PIN lesions, and 30 morphologically normal tissue samples are shown in Fig. 3 . The color scale ranges from yellow (no methylation) to black (100% methylation). The dendogram shows two major groups that we designated as "low methylation" and "high methylation." Interestingly, most normal prostate tissue samples (24 of 30 cases) clustered together in the low methylation group, whereas most prostate carcinomas and high-grade PIN lesions clustered together in the high methylation branch (86.7% and 64.1%, respectively). However, a detailed analysis showed that high-grade PIN lesions from different patients generally clustered together and did not segregate closely with the respective index tumor. When cluster analysis was applied to prostate carcinomas separately, it was apparent that all low methylation cases were organ-confined tumors (pT2a or pT2b) with Gleason score 5 and 6 (Fig. 4 ), although these associations did not reach statistical significance.


Figure 3
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  		FIGURE 3. Dendogram generated from hierarchical cluster analysis of all samples. Bottom, color scale; top, three genes analyzed; right, sample type (prostate carcinoma, high-grade PIN, and normal prostate tissue).

 

Figure 4
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  		FIGURE 4. Dendogram generated from hierarchical cluster analysis of prostate cancer samples only. Bottom, gray scale; top, three genes analyzed; right, case ID, Gleason score, and pathologic stage of each tumor.

 
Methylation and Clinicopathologic Correlations
No correlation was found between PSA levels and tumor PMA of any of the three target genes. Interestingly, APC and GSTP1 PMA correlated positively with Gleason score (r = 0.55; P = 0.0016 and r = 0.39; P = 0.036, respectively). No correlation was found between pathologic stage and PMA for any of the target genes.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Patients and Methods
 References
 
The aim of this study was to determine the epigenetic heterogeneity of high-grade PIN lesions in an attempt to elucidate the timing of key epigenetic changes in prostate tumorigenesis. For that purpose, multiple high-grade PIN lesions from the same cancerous prostate and from different patients were collected and analyzed. Moreover, these lesions were compared with the respective index tumor and normal prostate tissue in an attempt to provide the basis of a hypothetical epigenetic progression model. The use of quantitative MSP allowed us to calculate the PMA in each lesion and outline the early occurrence of promoter methylation at RARß2 and APC followed by GSTP1 methylation in the progression to invasive cancer.

The overall frequencies of promoter methylation at the three genes examined in the current study are similar to those reported previously, with the notable exception of GSTP1 in high-grade PIN lesions (15, 17–20, 23, 24). In most of those reports, the frequency of high-grade PIN lesions with methylated GSTP1 promoter exceeded 40% and even reached 76.3% in a recent report from our group (20), contrasting with 28.1% found in the present study. The larger sample size (128 individual lesions), which constitutes the largest series of high-grade PIN thus far surveyed for epigenetic alterations, might partially explain this discrepancy. Moreover, the use of formalin-fixed paraffin-embedded tissue for laser capture microdissection, instead of fresh-frozen tissue used in previous studies, might account for a decrease in the quality of DNA, thus precluding the detection of low levels of GSTP1 promoter methylation in high-grade PIN lesions. Noticeably, we also found that most (60%) cancerous prostates carried at least one high-grade PIN lesion harboring GSTP1 hypermethylation. Thus, an additional explanation for these divergent results could reside in the selection of the high-grade PIN lesion in previous studies. Because no laser capture microdissection was used in most of those studies, including our own, it is likely that mainly large high-grade PIN lesions were microdissected, thus increasing the chance of detecting GSTP1 methylated alleles. At this stage, we do not know the frequency of methylation of high-grade PIN lesions from noncancerous prostates, although it is likely to be low according to the present results.

Because the main goal of the present study was to verify the epigenetic heterogeneity of high-grade PIN lesions and assess their similarity with the paired invasive adenocarcinoma, the frequency of methylation at a given CpG site is more informative than the overall frequency of methylation at the GSTP1 promoter. Moreover, the methylation of CpG sites analyzed at the GSTP1 promoter was shown previously to be correlated with gene silencing (25). Although the use of different sets of primers might increase the overall frequency of methylation at the GSTP1 promoter, as shown by Nakayama et al. (24), no methylation was detected in morphologically normal tissue in that particular study, whereas we were able to detect low levels of GSTP1 methylation. Moreover, the very same set of primers and probe were used in a previous report from our group and different results were obtained (20). Moreover, in a previous study of ours, we were able to detect GSTP1 promoter methylation as low as 3.16 genome equivalents using the same set of primers and probe (26). Hence, the quantitative MSP assay seems sufficiently sensitive for the purposes of our study. Indeed, high sensitivity and specificity is a key feature of fluorogenic real-time quantitative MSP, as this quantitative assay was shown to reliably detect promoter methylation in the presence of 10,000-fold excess of unmethylated alleles (i.e., a 10-fold increase in sensitivity over conventional MSP; ref. 27). Eventually, bisulfite sequencing would yield more information concerning the pattern of methylation at the GSTP1 promoter. However, it is unlikely that this methodology would provide informative results in most high-grade PIN lesions as the methylation levels were generally low (median, 0; interquartile range, 0/0.7) and direct sequencing is not sufficiently reliable for methylation detection when methylation levels are low (i.e., <25% at any one site; ref. 28). Finally, it should be acknowledged that the quantitative MSP assay used in the present study does not absolutely quantitate methylated alleles as the completely methylated target sequences will be more efficiently amplified than incompletely methylated sequences mainly at early PCR cycles. Thus, the actual PMA might be in fact greater than those determined by quantitative MSP.

The majority of the high-grade PIN lesions analyzed in our study were sufficiently distant from the invasive adenocarcinoma to dismiss the possibility of intraductal spread. This possibility was also minimized by the step sectioning of the prostate, which was done to obtain a fine map of the high-grade PIN lesions in each specimen. Although the complex architectural patterns that characterize high-grade PIN are not typical of intraductal spread, the histologic picture might not allow for an accurate discrimination between high-grade PIN and intraductal spread of invasive adenocarcinoma in some cases (29). Although there is no reliable method to accomplish this distinction at present (30), we think that our findings argue against the common occurrence of intraductal spread for two reasons. (a) If a significant proportion of the lesions interpreted as high-grade PIN were indeed intraductal spread by the invasive adenocarcinoma, a more close segregation of the various lesions in each patient would be apparent by hierarchical clustering analysis. (b) We show that the pattern and extent of epigenetic changes of high-grade PIN was in most patients intermediate between normal prostate tissue and invasive prostate adenocarcinoma, something that fits better with a role for high-grade PIN as a precursor lesion rather than resulting from intraductal extension of an invasive adenocarcinoma.

The current study confirmed our previous observation of a positive correlation between APC and GSTP1 PMA and Gleason grade (20). Interestingly, we also reported that prostate carcinomas displaying low methylation levels usually correspond to organ-confined and intermediate histologic grade disease (20). Thus, these groups of tumors deserve further study and evaluation after long-term follow-up in a larger series of patients. If true, quantitative MSP might serve as an important tool for the identification of prostate carcinomas with different aggressive potential that would benefit from appropriate therapeutic approach.

We found that normal prostate tissue samples frequently carried APC and RARß2 hypermethylation, although the respective PMA values were considerably lower than those of high-grade PIN and prostate carcinoma. In contrast, GSTP1 promoter methylation was seldom observed in morphologically normal epithelial cells. Moreover, only a few of these normal prostate tissue samples clustered in the high methylation group. This finding was expected as promoter methylation at several genes in morphologically normal epithelium from other organs has been reported previously (31). Importantly, these results suggest that APC and RARß2 hypermethylation occur at a very early stage in prostate carcinogenesis, initially affecting a small subset of morphologically normal epithelial cells. Theoretically, the progressive accumulation of cells that carry methylated alleles of these cancer-related genes (plausibly resulting in a growth or survival advantage) could result in the development of preneoplastic lesions (e.g., high-grade PIN) and eventually overtly malignant lesions (i.e., prostate adenocarcinoma). Importantly, this hypothesis does not preclude the development of adenocarcinomas in prostate glands lacking precursor lesions, such as high-grade PIN (32).

In general, high-grade PIN lesions showed lower median PMA values for APC, GSTP1, and RARß2 compared with prostate carcinomas, although some individual lesions displayed PMA values in the same range as prostate carcinomas. Furthermore, a close segregation of paired prostate carcinomas and high-grade PIN lesions was not observed in all cases. Thus, a clonal relation between the index tumor and the high-grade PIN lesions found in the same prostatectomy specimen might not be apparent. If such prostate carcinomas arose from high-grade PIN, this preneoplastic lesion was most probably obliterated by the invasive neoplasia, accounting for its lack of representation. In this vein, it is noteworthy that the physical proximity between adenocarcinoma glands and high-grade PIN lesions should not be construed as definitive evidence of clonal relation as shown previously (22). However, the epigenetic similarity between some high-grade PIN lesions and most prostate carcinomas disclosed by cluster analysis (mainly the extreme of the high methylation group) might be interpreted as a measure of the potential of such preneoplastic lesions to evolve into an invasive carcinoma.

The wide variation observed in GSTP1, APC, and RARß2 methylation levels and the high frequency of methylation among high-grade PIN lesions might be explained by a "field effect." Hypothetically, one or several endogenous or exogenous carcinogenic insults could favor the epigenetic silencing of critical cancer-related genes in morphologically normal prostate cells leading to the development of several independent high-grade PIN lesions and eventually prostate carcinomas. The higher frequencies of methylation at the APC and RARß2 promoters as well as the increasing methylation levels observed from normal prostate tissue to high-grade PIN to prostate carcinomas indicate that clonal expansion of cells carrying methylated APC and RARß2 occurs early and in rapid succession. In the transition from normal prostate tissue to high-grade PIN, RARß2 promoter methylation probably occurs slightly earlier in most cases. On the other hand, GSTP1 methylation arises almost exclusively in high-grade PIN and only a small proportion of these lesions achieve the clonal expansion seen in prostate carcinomas. Indeed, whereas GSTP1 promoter methylation only arises in some high-grade PIN lesions and in a minority of morphologically normal prostate epithelial cells, it is common among prostate carcinomas. These finding suggest that GSTP1 hypermethylation is a marker associated with the progression to invasive carcinoma and provides a biological rationale for the reported high specificity of quantitative GSTP1 promoter methylation assays in molecular detection of prostate cancer (15, 20, 33). The role of GSTP1 silencing in favoring prostate cancer progression may be due to its facilitation of genomic damage. Indeed, the protein coded by the GSTP1 gene is involved in DNA protection from oxidants and electrophiles and prostate cells lacking GSTP1 expression become more susceptible to endure further genomic damage (34). The consequent accumulation of genomic alterations might then be responsible for the progression of established precancerous lesions. Despite the similar morphology, our results suggest that high-grade PIN lesions do not have equal potential for transformation into an invasive adenocarcinoma.

Thus, we have documented the epigenetic heterogeneity of high-grade PIN lesions at three gene promoters commonly methylated during prostate carcinogenesis. Aberrant APC and RARß2 promoter methylation are early events in this process and may contribute to the development of morphologically benign cell clusters with malignant potential. Finally, our findings suggest that higher levels of GSTP1 promoter methylation are associated with the transition from in situ lesions to invasive neoplasia and thus may serve as a marker of clinically important high-grade PIN lesions.


   Patients and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
References
 
Patients, Sample Collection, and DNA Extraction
We prospectively collected 30 consecutive radical prostatectomy specimens from patients with clinically localized prostate adenocarcinoma [stages T1c and T2 according to the tumor-node-metastasis staging system (35)] that harbored at least one high-grade PIN lesion. These patients were diagnosed and primarily treated at the Portuguese Oncology Institute-Porto (Porto, Portugal). The specimens were formalin fixed for 24 hours and processed for paraffin embedding. From each tissue block, a series of four 5-µm-thick sections were cut. The first section was H&E stained for pathologic evaluation: identification of the index or dominant tumor [assessed for Gleason grade (36) and tumor-node-metastasis stage (35)], high-grade PIN lesions [according to the criteria defined by the 1989 International Workshop on Prostatic Intraepithelial Neoplasia (4, 37)], and morphologically normal prostate tissue (without atrophy or inflammation) from the peripheral region (normal prostate tissue) distant (at least 1 cm) from neoplastic lesions. Because low-grade PIN is not reproducibly distinguishable from normal or hyperplastic epithelium (38), it was not included in this study. Foci of high-grade PIN were considered separate when they were >2 mm apart (7, 9). Then, target epithelial cells from the corresponding areas were procured by laser capture microdissection using a Pixcell LCM system (Arcturus Engineering, Inc., Mountain View, CA). An average of 400 laser shots (30 µm shot size, 60 ms laser pulse duration, and power of 60 MW) were used for each sample. Microdissected cells were then incubated overnight at 37°C in 50 µL digestion buffer [10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 1% Tween 20, 1 mg/mL proteinase K] and incubated at 95°C for 10 minutes to inactivate the proteinase K.

Relevant clinical data were collected from patients' clinical records. These studies were approved by the institutional review board of Portuguese Oncology Institute-Porto. Permission to test these samples without identifiers was also granted by exemption from the institutional review board of Johns Hopkins University.

Bisulfite Treatment and Quantitative MSP
Each DNA sample (50 µL) was used for sodium bisulfite modification as described previously (39). The sodium bisulfite reaction converts unmethylated (but not methylated) cytosine residues to uracil. Modified DNA was then purified using the Wizard purification resin (Promega, Madison, WI), treated again with NaOH, precipitated with ethanol, resuspended in 60 µL water, and stored at –80°C.

The modified DNA was used as a template for real-time fluorogenic MSP. The primers and probes used for APC, GSTP1, and RARß2 are described elsewhere (15, 19, 40). In addition, primers and a probe were used to amplify areas without CpG nucleotides of ACTB, an internal reference gene (41). The relative degree of methylation of each target gene in a given DNA sample (i.e., the PMA) was calculated according to the method described previously (42).

Fluorogenic quantitative MSP assays were carried out in a reaction volume of 20 µL in 384-well plates in an Applied Biosystems 7900 Sequence Detector (Perkin-Elmer, Foster City, CA). PCR was done in separate wells for each primer/probe set and each sample was run in triplicate. The final reaction mixture consisted of 600 nmol/L of each primer (Invitrogen, Carlsbad, CA); 200 nmol/L probe (Applied Biosystems, Foster City, CA); 1 unit Amplitaq Gold DNA polymerase (Perkin-Elmer); 200 µmol/L each of dATP, dCTP, dGTP, and dTTP; 5.5 mmol/L MgCl2, 1x Taqman buffer A; and 5 µL bisulfite-converted genomic DNA. PCR was done using the following conditions: 95°C for 10 minutes followed by 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. Each plate included multiple water blanks, a negative control, and serial dilutions of a positive control for constructing the calibration curve on each plate. Leukocyte DNA collected from healthy individuals was used as negative control. The same leukocyte DNA was methylated in vitro with SssI bacterial methyltransferase (New England Biolabs, Inc., Beverly, MA) and used as positive control for all studied genes.

Hierarchical Clustering
To perform unsupervised hierarchical cluster analysis of the several prostatic lesions based on the promoter methylation data, the PMA for the three genes studied were registered in an Excel spreadsheet, saved as a tab-delimited text file, and loaded into the clustering software J-Express Pro 2.5 (Molmine, Bergen, Norway). The cluster method used was complete linkage with Euclidean distance metric. The dendograms thus obtained grouped the prostatic lesions with the highest degree of epigenetic similarity in short-branched clusters, with longer branches indicating increasing disparity. The clustering together of two lesions from the same patient indicated clonal relatedness among them, whereas the clustering of prostatic lesions from different patients signified that they had followed similar epigenetic pathways. Each type of sample (normal prostate tissue, high-grade PIN, and invasive carcinoma) was also analyzed separately to find associations for each tested gene promoter.

Statistical Analysis
For each gene, the frequency of methylated and unmethylated cases as well as the median and interquartile range of the PMA for each group of tissue samples were determined. The Shapiro-Wilk's W test allowed for the examination of the appropriateness of a normal distribution assumption for each of the variables (data not shown). Then, values were analyzed using nonparametric tests (i.e., the Kruskal-Wallis one-way ANOVA) followed by the Bonferroni-adjusted Mann-Whitney U test when appropriate. For this comparison test among the three groups of tissue samples, the nonadjusted statistical level of significance of P < 0.05 corresponds to a Bonferroni-adjusted statistical significance of P < 0.0167. The correlations between the tumor PMA on the one hand and PSA level, Gleason score, and pathologic stage on the other were determined by calculating a Spearman correlation coefficient. The correlation between pathologic stage or tumor grade and the level of tumor methylation (i.e., low methylation versus high methylation) was assessed by the {chi}2 test or Fisher's exact test when appropriate. Statistical analyses were carried out using a computer-assisted program (Statistica for Windows version 6.0, StatSoft, Tulsa, OK). All statistical tests were two sided.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
Grant support: NIH grant U01CA84986-04 entitled "Integrated Development of Novel Molecular Markers—The Early Detection Research Network: Biomarkers Developmental Laboratories"; Liga Portuguesa Contra o Cancro-Núcleo Regional do Norte, Portugal (R. Henrique); Fundação para a Ciência e a Tecnologia (POCTI/CBO/38853/2001 and Projecto de Investigação 03-05 do Centro de Investigação-IPO Porto), Portugal (C. Jerónimo); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (BEX 21303-7), Brazil (A.L. Carvalho).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: R. Henrique and C. Jerónimo contributed equally to this work and should be regarded as joint first authors.

This article is affectionately dedicated to Manuel Jerónimo-Henrique.

Received 7/27/05; revised 11/ 8/05; accepted 12/ 5/05.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 

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