Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a detailed study using laser-capture microdissection

Abstract

Somatic inactivation of the glutathione S-transferase-pi gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in approximately 70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-alpha (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial.

Figure 1.

Promoter region of the GSTP1 gene. Two different primer sets targeting densely methylated alleles are depicted. Both primer sets have a common reverse primer.

Figure 2.

Histology of PIA of the prostate. A: Low-power view of PIA (dotted lined area) occurring adjacent to adenocarcinoma (outlined area) occupied a very large percentage of the total peripheral zone (PZ). A hyperplasia nodule (BPH) is seen in transitional zone (TZ). B: Intermediate-power view of boxed area. This lesion was classified as simple atrophy (SA). Original magnifications: ×12.5 (A); ×40 (B).

Figure 3.

Immunohistochemical features of PIA. A: Medium-power view of PIA stained with H&E. Asterisk indicates adenocarcinoma. B: Medium-power view of PIA stained with the 34βE12 monoclonal antibody. C: Heterogeneity of expression of GSTP1 in PIA. D: Higher power view of area in part of C. In parts B and D, arrows indicate positive epithelial cells in PIA, and arrowheads indicate PIA cells with no staining. Original magnifications: ×100 (A–C); ×200 (D).

Figure 4.

Atrophic gland showing features of low-grade PIN. Region of PIA showing scattered cells with slight nuclear enlargement and prominent nucleoli (arrows) designated as PIA containing focal low-grade PIN. Original magnifications: ×200; ×600 (inset).

Figure 5.

Example of tissue sections scanned with the BLISS system before and after LCM. A–D: Mixed simple atrophy and postatrophic hyperplasia lesion from a representative case was successfully microdissected where the same slide was scanned before and after microdissection using the BLISS system. Arrows indicate large acini classified as simple atrophy and arrowheads indicate small round acini classified as postatrophic hyperplasia. C and D: A separate case showing areas of atrophic epithelium merging directly with epithelium containing more cytoplasm and nuclear atypia (high-grade PIN). Note in D that high-grade PIN cells have not been procured. Original magnifications: ×25 [A (before LCM), B (after LCM)]; ×200 [C (before LCM), D (after LCM)].

Figure 6.

PIA merging with adenocarcinoma (CaP). A: Low-power view of a focus of PIA apparently merging with prostatic adenocarcinoma stained with H&E. Arrows indicate PIA lesion. Arrowheads indicate adenocarcinoma acini. B: Medium-power view of area in A. C: High-power view. D: High-power view of region of atrophy apparently merging with adenocarcinoma stained with the 34βE12 monoclonal antibody. Arrowheads indicate adenocarcinoma acini. Asterisks indicate the lumen of atrophic acinus. Original magnifications: ×40 (A); ×100 (B); ×400 (C, D).

Figure 7.

MSP of GSTP1 in prostate tissues. The tumor cell line LNCaP as a positive control for GSTP1 methylation, normal human tonsil DNA as a negative control, and a water control for contamination in the PCR reaction are indicated. DNA size markers are present at the ends of each gel. Lanes designated M, amplified products with GSTP1-methylated primer 1; lanes designated U, amplified products with GSTP1 unmethylated primer; lanes designated G, amplified products with GSTP1-methylated primer 2; lanes designated D, amplified product with MYOD1 primer. Top: The two panels are from patient 12 where microdissected tissues were obtained from areas of normal, BPH, PIA (two separate lesions), high-grade PIN (HGPIN), and adenocarcinoma (CaP). The cancer lesion (CaP) from patient 12 shows methylation with methylated primer set 1 and the high-grade PIN (HGPIN) lesion from patient 12 shows methylation with methylated primer set 2. Other tissues from patient 12 showed no evidence of methylation with either primer set. Bottom: The two panels are from patient 25. In the top portion, methylation was detected in the CaP lesion, and in the bottom portion, methylation was detected in the second PIA lesion as well as the HGPIN and CaP lesions. Only the cancer lesion in patient 25 showed methylation with both primer sets.