High-grade Prostatic Intraepithelial Neoplasia Lesions Research Articles

PTEN genomic deletion is an early event associated with ERG gene rearrangements in prostate cancer

To investigate the interaction between, and significance of, ERG gene rearrangements and PTEN genomic deletions in relation to the development and progression of prostate cancer (PCA). We interrogated an initial cohort of 220 men with localized PCA using fluorescence in situ hybridization for ERG rearrangements and PTEN genomic deletions. The incidences of ERG rearrangements and PTEN deletions in PCA were significantly higher than in high-grade prostatic intra-epithelial neoplasia (HGPIN) and benign prostate tissue (P < 0.001). ERG rearrangements and PTEN deletions were detected in 41.9 and 42.6% of patients' tumours, respectively. ERG rearrangements were never detected in benign prostate tissue, while PTEN aberrations were present at a basal level of 4.6%. PTEN hemizygous deletions showed higher frequency than homozygous deletions within each diagnostic category from benign prostate tissue to HGPIN and PCA (P ≤ 0.001). Furthermore, in 29 patients where all three tissues were available, PTEN genomic aberrations in PCA were significantly different from those in benign tissue (P = 0.005) and HGPIN (P = 0.02), reflecting the accumulation of genomic aberrations in the early stages of disease progression. Within this cohort, 71.4% of homozygous and 44.2% of hemizygous PTEN deletions occurred simultaneously with ERG rearrangements (P ≈ 0). Stratified according to Gleason score (GS), hemizygous PTEN deletions across various GS groups were observed at a higher frequency than homozygous deletions. However, PTEN homozygous deletions showed positive trends with higher GS, increasing in poorly differentiated PCA (GS 8-10) in comparison to moderately and well differentiated tumours (GS 6 and 7). We show significant association between ERG gene rearrangements and PTEN genomic aberrations in subset of PCA. Our analysis also provides further support for the observation that homozygous PTEN deletions can occur within the subset of HGPIN lesions, and shows accumulating genetic aberrations with disease progression, evidenced by higher detection in PCA than in HGPIN and more PTEN homozygous deletions in GS 8-10 than in 6-7.

Expression analysis of putative stem cell markers in human benign and malignant prostate

Stem cells were suggested to be present in human prostate cancer as a small population of distinct cells, which may contribute to carcinogenesis, tumor recurrence, and chemoresistance. To identify potential prostatic stem cells, we analyzed the expression of several potential stem cell markers in benign prostate and prostatic adenocarcinoma. CD44, CD133, Oct4, SOX2, and EZH2 expression was detected by immunohistochemical (IHC) staining using tissue microarray assays (TMA) composed of benign (non-neoplastic) prostatic tissue, high grade prostatic intraepithelial neoplasia (HGPIN), and prostatic adenocarcinoma. Positive staining was defined as 1+ (<10%), 2+ (10-50%), or 3+ (>50%). We found CD44 staining in 97% and 72% of benign + HGPIN and malignant lesions, respectively. CD133 staining was detected in a small fraction (4 of 67) of prostate carcinomas. We found that Oct4 nuclear expression was strongly associated with benign lesions and HGPIN but not prostate cancer (P < 0.05). In most cases, nuclear expression of EZH2 and SOX2 was detected in less than 10% of cells in non-neoplastic prostate glands, HGPINs or prostate adenocarcinomas. Moreover, 27 of 33 SOX2 1+ prostate cancers were also EZH2 1+, whereas all 33 of these cases were CD44+. Expression of CD44 and Oct4 identified large populations of benign and malignant cells in the prostate, which did not fit the definition of stem cells as a small fraction of the total cell population. Our results suggest that combined expression of embryonic stem cell markers EZH2 and SOX2 might identify potential cancer stem cells as a minor (<10%) subgroup in CD44+ prostatic adenocarcinoma.

Abstract 5681: Flavokawain B down-regulates SKP2 through a proteasome-dependent and ubiquitin-independent pathway, and acts synergistically with Bortezomib on inhibition of Skp2 expression and prostate cancer cell growth

Abstract The E3 ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), an oncogenic protein, was found to be overexpressed in 557 of 622 (89.2%) primary prostate cancer (PCa) samples and in 64 of 74 samples (86.4%) high-grade prostatic intra-epithelial neoplasia lesions. In addition, a transgenic mouse model with prostatic over-expression of SKP2 develops low-grade adenocarcinomas. These results suggest that SKP2 could be a potential target for PCa chemoprevention. In a search for SKP2 targeting agents for PCa chemoprevention, we found that Flavokawain B, a chalcone found in multiple edible plants- including roots of Alpinia speciosa (family Zingiberaceae) and Piper methysticum Forst (kava kava), potently down-regulates the protein expression of SKP2 in several prostate cancer cell lines (LNCaP, C4-2B, DU145 and PC3) at a concentration of 8.8 µM. More importantly, Flavokawain B reduces the cell viability of transfected PC3 cell line stably overexpressing SKP2 compared to vector control-transfected PC3 cells (IC50s are 7 and 26.4 µM, respectively), which suggest that flavokawain B may target SKP2 for its growth inhibitory effect. Flavokawain B treatment did not decrease the promoter activity and mRNA levels of SKP2. Instead, Flavokawain B decreases the protein levels of both a native and a myc-tagged SKP2 in PC3 cells through promoting its degradation. The degradation of the myc tagged SKP2 can be recovered by a proteasome inhibitor MG132. MG132 treatment resulted in an increase of ubquitinated SKP2, whereas Flavokawain B did not affect SKP2 ubquitination. These data suggest that the degradation of SKP2 by Flavokawain B may be through a proteasome-dependent and ubquitination-independent pathway. Interestingly, proteasome inhibitors (MG132 and Bortezomib) also down-regulate SKP2 mRNA expression through transcriptional inhibition, and combination of Bortezomib or MG132 with Flavokawain B exhibits a synergistic effect on SKP2 down-regulation and cell growth inhibition via induction of G2M arrest in PCa cell lines. Taken together, Flavokawain B is a novel SKP2 target agent and deserves further investigation for PCa chemoprevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5681.

Value of magnetic resonance spectroscopy (MSR) and dynamic contrast-enhanced magnetic resonance (DCEMR) imaging for the characterization of high-grade prostatic intraepithelial neoplasia (HGPIN) foci

Background Despite an increasing interest in high-grade prostatic intraepithelial neoplasia (HGPIN), the clinical suspicious aspect of this premalignant lesion remains poorly characterized. The aim of this study was to analyze the magnetic resonance spectroscopy (MSR) and dynamic contrast-enhanced magnetic resonance (DCEMR) imaging features of isolated HGPIN lesions. Materials and methods From January 2007 to January 2009, 330 cases were included in a protocol that involve the use of MSR and DCEMR for the diagnosis of prostate diseases. Of these, 27 patients with isolated (no associated prostate cancer diagnosis) HGPIN histologic diagnosis at the first prostate biopsy were included in the present study. All cases were previously submitted to MSR/DCEMR (1.5 T scanner) and, no later than 10 days to a random 12-core biopsy scheme. Biopsy targeting was done in zones corresponding to those analyzed with MSR and DCEMR. Results In the 27 patients, 30 HGPIN foci with a diameter of 6 mm or greater were analyzed and compared with 27 peripheral zone areas of normal prostate tissue. With MSR, HGPIN foci were characterized by a significantly higher ( P < 0.05) absolute value of choline and choline + creatine/citrate ratio compared with normal tissue. With DCEMR, HGPIN foci were characterized by lower values of all dynamic parameters but differences did not reach statistical significance ( P > 0.05). Conclusions In our experience, HGPIN lesions can be metabolically characterized by MSR through the absolute value of choline and the choline + creatine/citrate ratio.

POD-04.05: Detection of TMPRSS2-ERG and TMPRSS2-ETV1 Fusion Genes in Prostate Cancer and Benign Prostate Hyperplasia

Recent studies have reported that a high proportion of prostate cancers express fusion genes. These fusion genes occur when the 5' region of the androgen regulated TMPRSS2 gene merges with one of the ETS family transcription factors, mainly ERG and rarely ETV1 and ETV4. The presence of these fusions has been tested in a subset of high grade PIN lesions and in a small number of benign prostatic hyperplasia samples.

Simultaneous haploinsufficiency of Pten and Trp53 tumor suppressor genes accelerates tumorigenesis in a mouse model of prostate cancer

Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.

Characterization of TMPRSS2-ERG Fusion High-Grade Prostatic Intraepithelial Neoplasia and Potential Clinical Implications

More than 1,300,000 prostate needle biopsies are done annually in the United States with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer on subsequent needle biopsies in prostate-specific antigen-screened populations. In contemporary series, prostate cancer is detected in approximately 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histologic subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in prostate cancer has also been shown to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive clinical course. In this study, we assessed a series of HGPIN lesions and paired prostate cancer for the presence of TMPRSS2-ERG gene fusion. Fusion-positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances, the matching cancer shared the same fusion pattern. Sixty percent of TMPRSS2-ERG fusion prostate cancer had fusion-negative HGPIN. Given the more aggressive nature of TMPRSS2-ERG prostate cancer, the findings of this study raise the possibility that gene fusion-positive HGPIN lesions are harbingers of more aggressive disease. To date, pathologic, molecular, and clinical variables do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion prostate cancer in subsequent biopsies.

PTOV1 Expression Predicts Prostate Cancer in Men with Isolated High-Grade Prostatic Intraepithelial Neoplasia in Needle Biopsy

To analyze the expression of PTOV1 in high-grade prostatic intraepithelial neoplasia (HG-PIN) and to explore its usefulness to predict prostate cancer in patients with isolated HG-PIN in needle biopsy (prostate needle biopsy). PTOV1 expression in HG-PIN lesions from 140 patients was analyzed by immunohistochemistry in a semiquantitative manner (Histo-score). HG-PIN derived from 79 radical prostatectomies for prostate cancer and from 11 cistoprostatectomies for bladder cancer without prostate cancer were used as positive and negative controls, respectively. Fifty patients with HG-PIN without concomitant cancer at their first prostate needle biopsy were chosen as the study group. Patients were followed by a mean of 2.5 repeated prostate needle biopsies (1-5), during a mean period of 12.4 months (1-39). PTOV1 expression in HG-PIN from radical prostatectomies showed a significantly higher Histo-score (162.6) compared with specimens from cistoprostatectomies (67.0). In the study group, PTOV1 expression was significantly higher in samples with cancer in the follow-up (11 patients, 22%) compared with samples in which cancer was not detected (151.4 versus 94.6). PTOV1 expression was the only independent predictor of cancer in the multivariate analysis and the area under the curve was 0.803 (95% confidence interval, 0.728-0.878). A threshold of 100 for PTOV1 expression provided 90.9% sensitivity, 51.3% specificity, 34.5% positive predictive value, and 95.2% negative predictive value. PTOV1 is overexpressed in HG-PIN associated with cancer and is a potential marker for studying the carcinogenesis and progression of prostate cancer. Prostate needle biopsy with PTOV1 expression in HG-PIN above a threshold of 100 should be repeated immediately for the likely presence of undiagnosed cancer.

Decreased NKX3.1 protein expression in focal prostatic atrophy, prostatic intraepithelial neoplasia, and adenocarcinoma: Association with Gleason score and chromosome 8p deletion: Bethel CR, Faith D, Li X, Guan B, Hicks JL, Lan F, Jenkins RB, Bieberich CJ, De Marzo AM, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Baltimore, MD

NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC), and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in 5 of 7 cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease.

Functional Analysis of the Aurora Kinase A Ile31 Allelic Variant in Human Prostate

Overexpression of the centrosome-associated serine/ threonine kinase Aurora Kinase A (AURKA) has been demonstrated in both advanced prostate cancer and high-grade prostatic intraepithelial neoplasia lesions. The single-nucleotide polymorphism T91A (Phe3lile) has been implicated in AURKA overexpression and has been suggested as a low-penetrance susceptibility allele in multiple human cancers, including prostate cancer. We studied the transcriptional consequences of the AURKA Ile31 allele in 28 commercial normal prostate tissue RNA samples (median age, 27 years). Significant overexpression of AURKA was demonstrated in homozygous and heterozygous AURKA Ile31 prostate RNA (2.07-fold and 1.93-fold, respectively; P < .05). Expression levels of 1509 genes differentiated between samples homozygous for Phe31 alleles and samples homozygous for Ile31 alleles (P = .05). Gene Ontology classification revealed overrepresentation of cell cycle arrest, ubiquitin cycle, antiapoptosis, angiogenesisrelated genes. When these hypothesis-generating results were subjected to more stringent statistical criteria, overexpression of a novel transcript of the natural killer tumor recognition sequence (NKTR) gene was revealed and validated in homozygous Ile31 samples (2.6-fold; P < .05). In summary, our data suggest an association between the AURKA Ile31 allele and an altered transcriptome in normal non-neopasic prostates.

Upregulation of erythropoietin receptor in human prostate carcinoma and high-grade prostatic intraepithelial neoplasia

The aim of the present study was to investigate the differential expression of erythropoietin receptor (EPOR) in prostate carcinoma (PCa), high-grade prostatic intraepithelial neoplasia (PIN), prostatic hyperplasia (BPH) lesions and normal prostatic tissues by immunohistochemistry; and to test the hypothesis that upregulation of EPOR is a specific event for prostate carcinogenesis. An immunohistochemical analysis of EPOR was performed on 30 PCa, 50 BPH with/without inflammation lesions and 30 normal prostatic tissue samples. EPOR staining was quantitated and classified into normal expression and overexpression. Totally 16 high-grade PIN lesions were found in this study. Overexpression of EPOR was shown only in PCa and high-grade PIN. Statistical analysis demonstrated that higher median EPOR staining score of PCa and high-grade PIN in comparison with BPH (P < 0.05) and higher median EPOR staining score of PCa compared with high-grade PIN (P < 0.05). Our data demonstrate that upregulation of EPOR is not uncommon for PCa and upregulated EPOR in high-grade PIN suggests upregulation of EPOR is an early event for prostate carcinogenesis. The role of upregulated EPOR and possibly enhanced EPOR signaling in prostate carcinogenesis warrants further studying.

Epigenetic events, remodelling enzymes and their relationship to chromatin organization in prostatic intraepithelial neoplasia and prostatic adenocarcinoma

To explore the nuclear chromatin phenotype, overall epigenetic mechanisms, chromatin remodelling enzymes and their role as diagnostic biomarkers in prostate lesions, using high-resolution computerized quantitative digital image analysis (DIA). A tissue microarray (TMA) was constructed using paraffin wax-embedded prostatic tissues from 78 patients, containing 30 cores of benign prostatic hyperplasia (BPH), 10 of low-grade prostatic intraepithelial neoplasia (LGPIN), 38 of prostate adenocarcinoma, 20 of BPH tissue excised at 0.6-1 mm from LGPIN lesions, and 10 of BPH prostatic tissues obtained 0.6-1 mm from high-grade PIN (HGPIN) lesions. Chromatin phenotype was assessed on haematoxylin-stained sections using high-resolution texture analysis. For quantitative immunohistochemistry, antibodies raised against acetylated histone H3 lysine 9 (AcH3K9), 5'methylcytidine (5MeC) and the chromatin remodelling ATPase ISWI (SNF2H and SNF2L) were used. The immunodensity was measured using DIA to determine the epigenetic profile of the cases. At least 60 nuclei were measured from each case. There were many statistically significant differences in staining intensity and nuclear distribution patterns in chromatin phenotype and immunostaining (p </= 0.001). These changes allowed the differentiation between the various pathological subgroups with a classification accuracy of 76-100% using chromatin phenotype or immunostaining measuring epigenetic and chromatin remodelling changes (5MeC, AcH3K9 and ISWI). In PIN lesions, there was a high chromatin content with DNA-hypermethylation, while in prostatic adenocarcinoma there was a lower chromatin content with DNA-hypomethylation and H3K9-hypoacetylation. There was significantly more ISWI protein in neoplastic tissues. There were malignancy-associated changes (MAC) in chromatin phenotype and overall epigenetic events in BPH tissues adjacent to PIN lesions. The present study confirms the ability of high-resolution computerized digital imaging of nuclear texture features to detect changes in chromatin phenotype, epigenetic events and the presence of chromatin remodelling, factors that can be used to distinguish between different prostatic pathologies, i.e. BPH, LGPIN, HGPIN and prostate adenocarcinoma, and further allow the detection of MAC near PIN lesions. These results provide a base for future diagnostic applications of DIA combined with immunohistochemistry. Our experiments underscore the importance of epigenetic mechanisms during carcinogenesis. Further studies are needed to elucidate the complex interplay between chromatin structure, its modifications and the progression of prostate cancer.

Relaxin Promotes Prostate Cancer Progression

To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. The relaxin and its receptor leucine-rich repeat containing G protein-coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. Relaxin signaling plays a role in prostate cancer progression.

Immunohistochemical Expression of Endothelin-1 and Endothelin-A and Endothelin-B Receptors in High-Grade Prostatic Intraepithelial Neoplasia and Prostate Cancer

ObjectivesTo analyze the expression of endothelin-1 (ET-1), endothelin-A receptor (ET-A-R), and endothelin-B receptor (ET-B-R) in incidental prostate cancer in cystoprostatectomies (CyPs), clinically detected hormonally untreated and hormonally treated prostate cancer in radical prostatectomies (RPs), and hormone-independent prostate cancer in transurethral resections of the prostate (TURPs). High-grade prostatic intraepithelial neoplasia (HGPIN) was also investigated. MethodsNineteen CyPs and 44 RPs (25 untreated, 19 treated) with pT2a Gleason score 6 cancer and HGPIN were examined. The study included 9 TURPs with hormone-independent cancer and 8 normal cases from CyPs without prostate cancer and HGPIN. ET-1, ET-A-R, ET-B-R, and the proliferation marker Ki67 were investigated immunohistochemically. ResultsThe mean proportion of prostate cancer cells with strong ET-1, ET-A-R, and ET-B-R expression in CyPs was lower (18.5%, 28.0%, and 14.7%, respectively) than in the untreated group (40.7%, 39.7%, and 25.1%) and higher than in treated group (5.0%, 13.9%, and 11.3%). The highest values were in the hormone-independent cancer group (53.9%, 48.9%, 33.3%). The trend in the proportion of HGPIN cells overexpressing ET-1, ET-A-R, and ET-B-R was similar to that in the cancer groups. The values in HGPIN lesions were always slightly greater than those in the cancers. Ki67 expression in HGPIN and prostate cancer in CyPs was lower than in RPs and TURPs. ConclusionsOur study showed for the first time that ET-1, ET-A-R, and ET-B-R expression is not limited to the late prostate cancer phases. It is also seen in HGPIN as well as in prostate cancers considered to be clinically insignificant, such as those seen in CyP specimens. Although the series of cases in each group was small, our data may have clinical significance.

High-Grade Prostatic Intraepithelial Neoplasia in Patients with Prostate Cancer: MR and MR Spectroscopic Imaging Features—Initial Experience

To retrospectively determine the magnetic resonance (MR) and MR spectroscopic imaging features of high-grade prostatic intraepithelial neoplasia (HGPIN) in patients with prostate cancer. Approval of the committee on human research was obtained, with a waiver of consent for this HIPAA-compliant study. Endorectal MR imaging and MR spectroscopic imaging were performed in 48 men (mean age, 59 years; range, 47-75 years) prior to radical prostatectomy for prostate cancer. T2-weighted signal intensity and metabolic ratios of peripheral zone HGPIN foci of 6 mm or greater in diameter were recorded by two readers with knowledge of step-section histopathologic findings using areas of confirmed normal and cancerous peripheral zone tissue for comparison. A random effects statistical model was used to compare metabolic ratios from normal, HGPIN, and cancer voxels. A total of 123 peripheral zone HGPIN foci with a mean diameter of 3 mm (range, 1-28 mm) were identified in 37 (77%) patients, but only 20 foci in 14 patients had a diameter of 6 mm or greater. Six foci were excluded, yielding 14 large HGPIN lesions from 11 patients in the final statistical analysis. The larger HGPIN foci were not associated with any focal reduction in T2-weighted signal intensity but demonstrated metabolic findings intermediate between normal and cancerous tissue; the mean ratios of choline (Cho) to creatine (Cr) for normal, HGPIN, and cancer were 0.92, 1.75, and 1.99, respectively, (P < .01), and the corresponding ratios of Cho plus Cr to citrate were 0.34, 0.50, and 0.78 (P < .01). HGPIN is metabolically intermediate between normal peripheral zone tissue and prostate cancer at MR spectroscopic imaging but does not manifest any MR imaging abnormality and is rarely of sufficient size to cause substantial error in evaluation of peripheral zone tumor extent in patients with prostate cancer.

Decreased NKX3.1 Protein Expression in Focal Prostatic Atrophy, Prostatic Intraepithelial Neoplasia, and Adenocarcinoma: Association with Gleason Score and Chromosome 8p Deletion

NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC) and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in five of seven cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease.

TMPRSS2-ERG Gene Fusion Causing ERG Overexpression Precedes Chromosome Copy Number Changes in Prostate Carcinomas, Paired HGPIN Lesions

TMPRSS2-ETS gene fusions have been found recurrently in prostate carcinomas, but not in the presumed precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN). However, HGPIN lesions may share chromosomal changes with prostate cancer. To determine the relative order of genetic events in prostate carcinogenesis, we have analyzed 34 prostate carcinomas, 19 paired HGPIN lesions, 14 benign prostate hyperplasias, 11 morphologically normal prostatic tissues for TMPRSS2-ERG, TMPRSS2-ETV1 rearrangements, genomic imbalances. TMPRSS2 exon 1 was fused in-frame with ERG exon 4 in 17 of 34 (50%) prostate carcinomas, in 4 of 19 (21%) HGPIN lesions, but in none of controls. The findings were further validated by sequencing analysis, by the real-time polymerase chain reaction quantification of TMPRSS2-ERG fusion transcript, the ERG exons 5/6:exons 1/2 expression ratio. Chromosome copy number changes were detected by comparative genomic hybridization in 42% of clinically confined carcinomas, in none of the 16 HGPIN lesions analyzed. We demonstrate for the first time that the TMPRSS2-ERG fusion gene can be detected in a proportion of HGPIN lesions, that this molecular rearrangement is an early event that may precede chromosome-level alterations in prostate carcinogenesis.

Study of microvessel density and the expression of the angiogenic factors VEGF, bFGF and the receptors Flt-1 and FLK-1 in benign, premalignant and malignant prostate tissues.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates endothelial cell growth and enhances vascular permeability. VEGF exerts its action by binding to the specific cell surface receptors, fms-like tyrosine kinase 1 (Flt-1) and fetal liver kinase 1 (FLK/KDR). In tumor angiogenesis, Vascular endothelial growth factor stimulates endothelial cells to produce Basic fibroblastic growth factor (bFGF), which further enhances angiogenic activity. Very little information on the expression of VEGF, bFGF, and the receptors Flt-1 and FLK/KDR is available. Herein, we evaluate the expression of these angiogenic factors and receptors in normal prostate, high grade prostate intraepithelial neoplasia (HGPIN) and prostatic cancer (CaP). 58 selected surgical specimens exhibiting areas of normal prostate, HGPIN, and CaP were evaluated for microvessel density, and for VEGF, bFGF, Flt-1 and FLK/KDR protein expression by immunohistochemistry. Results were correlated with pathological data. There was a statistically significant increase in the microvessel density and in the expression of the angiogenic factors VEGF, bFGF and the receptors FLK/KDR and Flt-1, in the premalignant and malignant tissues in comparison with normal prostatic glands. Microvessel density also correlated with higher Gleason grade, pathological stage and the expression of the receptors FLK/KDR and Flt-1. The "initiation switch" of angiogenesis was observed to be an early event consistent with the recruitment of new vasculature into high grade PIN lesions and it increased in the progression of prostatic cancer.

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

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 RARbeta2) 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 RARbeta2 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 RARbeta2 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.

The utility of Ki-67 expression in the differential diagnosis of prostatic intraepithelial neoplasia and ductal adenocarcinoma

Cribriform and/or papillary prostatic lesions observed on limited tissue, such as needle biopsy, can pose diagnostic dilemmas. One such area of difficulty is the distinction between papillary and/or cribriform prostatic high-grade prostatic intraepithelial neoplasia (HG-PIN) and ductal adenocarcinoma. Over 48 months, we identified 17 cases of ductal adenocarcinoma and 17 cases of HG-PIN from radical retropubic prostatectomy specimens. The HG-PIN lesions were in all cases associated with an acinar prostatic adenocarcinoma component. For each case, we evaluated the proliferative activity, assessed by Ki-67 immunohistochemistry. The majority (82%) of ductal adenocarcinomas were composed of mixed papillary and cribriform patterns, with the remaining demonstrating pure papillary or cribriform patterns. The HG-PIN lesions showed a papillary, cribriform, or mixed papillary/cribriform architecture. The proliferative activity, defined as Ki-67 labeling index, was statistically higher in ductal adenocarcinoma (mean 33%, range 21%-66%) as compared with HG-PIN (mean 6%, range 2%-15%), with no overlap in the Ki-67 indices (P = 0001). A combination of histological features and measurements of cellular proliferation may be helpful to distinguish HG-PIN from ductal adenocarcinoma in limited prostatic tissue samples.