A comparative study of tissue ω-6 and ω-3 polyunsaturated fatty acids (PUFA) in benign and malignant pathologic stage pT2a radical prostatectomy specimens.

OBJECTIVE: To analyze different polyunsaturated fatty acid (PUFA) tissue levels in malignant compared with benign prostatic tissue from the same prostate specimens. MATERIALS AND METHODS: Fresh frozen benign and malignant prostatic tissue was obtained from radical prostatectomy specimens in 49 men with pathologic stage pT2a prostate cancer. Histopathologic examination confirmed that all tissues from each prostate being analyzed were either completely benign or almost totally malignant. The PUFA composition in these tissues was determined by gas-liquid chromatography on a capillary column. The relative amount of each PUFA (% of total fatty acids) was quantified by integrating the area under the peak and dividing the result by the total area of all fatty acids. RESULTS: Tissue levels of dihomo-γ-linolenic acid, (C20:3w6), an ω-6 PUFA and a major precursor of ω-6 PUFA metabolites, were significantly higher in malignant compared with benign tissues (P = 0.002). Tissue levels of the downstream ω-6 metabolites, arachidonic acid (AA) (20:4ω6), and adrenic acid, (22:4ω6), were significantly lower in cancer tissues, (P < 0.0001 and P = 0.013, respectively). Overall, the total levels of ω-6 PUFA were lower in cancer (P = 0.001). CONCLUSION: We found that the ω-6 PUFA AA and adrenic acid are decreased in malignant prostatic tissues compared with benign tissues from the same prostates. These findings provide additional evidence that dietary fat is associated with prostatic carcinogenesis.

Endothelin-1 expression in prostate cancer and high grade prostatic intraepithelial neoplasia.

OBJECTIVE: To investigate the prognostic value of endothelin-1 (ET-1), a vasoconstrictor involved in differentiation and growth of cancer, in prostate cancer. STUDY DESIGN: A tissue microarray was constructed of 287 prostate cancers from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. Slides were immunostained for ET-1. Intensity and extent of immunoreactivity and their product (IRp) were evaluated. We separately arrayed benign prostatic tissue, atrophy, high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer from 40 men. RESULTS: ET-1 expression was stronger in both HGPIN and cancer than in benign tissue (p < 0.001). The intensity and the IRp of ET-1 predicted biochemical recurrence (p < 0.001 and p = 0.044, respectively), while the staining extent showed no significant correlation with outcome (p = 0.68). Recurrence-free survival in patients with strong ET-1 staining was shorter than in those with weaker expression (hazard ratio [CI 95%] 2.44 [1.55-3.84], p < 0.001). In a multivariate analysis including ET-1 expression, preoperative serum prostate-specific antigen (PSA), extraprostatic extension, margin status, seminal vesicle invasion and Gleason score, only PSA, margins and Gleason score remained significant. CONCLUSION: ET-1 is overexpressed in localized prostate cancer and predicts outcome after RP in univariate analysis.

Expression of redox pathway enzymes in human prostatic tissue.

OBJECTIVE: To evaluate the involvement of thioredoxin reductase (TxnR), thioredoxin (Trx) and peroxiredoxins (Prdx) in prostate cancer (PCa) and to assess the potential prognostic importance of these redox-regulated pathways. STUDY DESIGN: Expression of the isoforms TxnR2, Trx1 and Prdx2 was studied by immunohistochemistry on tissue microarrays (TMAs). In a prognostic TMA, 294 primary cases of PCa with a median follow-up of 49 months were stained for Trx1 and Prdx2. Another TMA containing benign prostatic tissue, atrophy, high grade prostatic intraepithelial neoplasia (HGPIN) and PCa from 40 patients was stained with all 3 antibodies. RES ULTS: All 3 proteins showed similar expression patterns, with the highest immunoreactivity in HGPIN followed by atrophy, PCa and benign tissue. TxnR2, Trx1 and Prdx2 were overexpressed in HGPIN and PCa compared with benign tissue (p < 0.001), and Trx1 and Prdx2 were also overexpressed in HGPIN compared with PCa (p < 0.001). Trx1 and Prdx2 did not correlate with biochemical recurrence. CONCLUSION: This study demonstrates up-regulation of the redox pathway proteins in PCa and its precursor lesions. The pathogenetic role of the redox system remains to be investigated.

Expression of PDX-1 in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic tissue.

Pancreatic duodenal homeobox 1 (PDX-1), a Hox type transcription factor, is necessary for differentiation of exocrine and endocrine pancreas, and regulates insulin gene transcription. PDX-1 expression was studied by immunohistochemistry on a tissue microarray (TMA) of 289 primary prostate cancers (PCa) from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. We separately arrayed benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and PCa from 40 men and also 17 lymph node metastases. Intensity and extent of immunoreactivity and their product (IRp) were evaluated by two independent observers. PDX-1 was overexpressed in cancer vs benign tissue (p<0.001), but also in atrophy and HGPIN vs cancer (p<0.001 and p=0.022, respectively). PDX-1 expression did not correlate with biochemical recurrence, but decreased with higher Gleason pattern (p<0.001) and in metastases vs primary PCa (p<0.001). Weighted kappa for interobserver agreement of intensity, extent and IRp was 0.65, 0.13 and 0.54, respectively. Presence of PDX-1 protein in benign and malignant prostatic tissue was confirmed by Western blot. In view of recent attention to the role of insulin systems in men with PCa, this protein is of interest in the pathogenesis of PCa.

Heat shock proteins 27, 60 and 70 as prognostic markers of prostate cancer.

Heat shock proteins (HSPs) protect cells against stress-associated injury and are overexpressed in several malignant tumors. We aimed to investigate their value as prognostic markers in prostate cancer. A tissue microarray (TMA) was constructed of 289 prostate cancers from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. Slides were immunostained for HSP27, HSP60 and HSP70. Intensity and extent of immunoreactivity (IR) and their product (IRp) was evaluated by two observers. The IRp of HSP27 and HSP60, but not of HSP70, significantly predicted biochemical recurrence (p=0.014, 0.034 and 0.160, respectively). Recurrence-free survival in patients with strong HSP27 and HSP60 staining was shorter than in those with weak expression (p=0.019 and 0.001, respectively). IRp of HSP27 and HSP60 correlated with Gleason score (p<0.01). HSP60 was an independent predictor of biochemical recurrence in multivariate analysis, including extraprostatic extension, margin status, seminal vesicle invasion and Gleason score. Weighted kappa for interobserver agreement of HSP27, HSP60 and HSP70 IR was 0.613-0.823 for intensity and 0.584-0.719 for IRp, but only 0.036-0.244 for extent, raising the question whether staining extent should be estimated on TMA. We conclude that HSP27 and HSP60 are predictors of biochemical recurrence after RP.

Prevalence of prostate cancer at different levels of serum prostate-specific antigen (PSA) and different free: total PSA ratios in a consecutive series of men referred for prostate biopsies.

OBJECTIVE: In this retrospective study we report on the detection rate of prostate cancer (PCa) at different levels of prostate-specific antigen in serum (s-PSA) and at different PSA ratios (free:total PSA) during a 2-year period in patients without previously known PCa. MATERIAL AND METHODS: During the years 2001 and 2002, 361 consecutive patients were examined with ultrasound-guided core needle biopsies at our department. The patients were biopsied due to an increased s-PSA level, a low PSA ratio or findings at digital rectal examination (DRE). Patients with previously known cancer (T1a/b or cancer already detected with fine-needle aspiration cytology) were excluded. We used the BioPince biopsy needle, which has a stroke length of 32 mm. In 91% of the patients, eight biopsies were taken from the apex, mid-medial, mid-lateral and base positions bilaterally. RESULTS: Of the 361 patients, 188 (52%) had PCa. Most cancers were T1c or T2 tumors (51% and 34%, respectively). Among patients with an s-PSA level of < 4 ng/ml, 8/35 (23%) had PCa. Five of 13 patients with a normal DRE (T1c) and an s-PSA level of < 4 ng/ml had PCa. In total, in the PSA ratio intervals 0.05-0.1 and 0.11-0.17, cancer was found in 71% and 51% of cases, respectively. In contrast, only 35% of patients had positive biopsies when the PSA ratio was normal (p<0.01). CONCLUSIONS: The overall cancer detection rate was high and a large proportion of patients with an s-PSA level of < 4 ng/ml had PCa. The risk of having PCa increased considerably with a low PSA ratio.

Long-term fatty fish consumption and renal cell carcinoma incidence in women.

CONTEXT: The epidemiological evidence that fatty fish consumption may be associated with the lower risk of several cancers is not consistent and no studies of renal cell carcinoma (RCC) exist. OBJECTIVE: To examine the association between fatty and lean fish consumption and risk of RCC in women. DESIGN, SETTING, AND PARTICIPANTS: The Swedish Mammography Cohort, a population-based prospective cohort study of 61,433 women aged 40 to 76 years without previous diagnosis of cancer at baseline (March 1, 1987-December 14, 1990). Participants filled in a food frequency questionnaire at baseline and in September 1997. MAIN OUTCOME MEASURE: Incident renal cell carcinoma. RESULTS: During a mean of 15.3 years (940,357 person-years) of follow-up between 1987 and 2004, 150 incident RCC cases were diagnosed. After adjustment for potential confounders, an inverse association of fatty fish consumption with the risk of RCC was found (P for trend = .02), but no association was found with lean fish consumption. Compared with no consumption, the multivariate rate ratio (RR) was 0.56 (95% confidence interval [CI], 0.35-0.91) for women eating fatty fish once a week or more. Compared with women consistently reporting no fish consumption, the multivariate RR was 0.26 (95% CI, 0.10-0.67) for those women reporting consistent consumption of fatty fish at baseline and 1997 (based on a subset of 36 664 women who filled in the baseline and 1997 questionnaires, with 40 incident RCC cases during the 1998-2004 follow-up period). CONCLUSION: Our study suggests that consumption of fatty fish may reduce the occurrence of RCC in women.

Cytological features of prostatic intraepithelial neoplasia.

Fine-needle aspiration cytology (FNAC) is an acknowledged method for diagnosing prostate cancer. False-positive results are uncommon, but concerns have been raised that prostatic intraepithelial neoplasia (PIN) could be misinterpreted as carcinoma. Therefore, we attempted to describe cytological features of PIN. Cells were scraped from macroscopically normal areas of 177 radical prostatectomy specimens, smeared and Giemsa-stained. Histological slides from these areas were reviewed, and 17 samples with high-grade PIN and with no invasive cancer were selected. Smears from 17 invasive cancers were used for comparison. Cancer showed high cellularity and dissociation, while PIN smears only contained a few clusters of atypical cells. Pronounced nuclear atypia, prominent or multiple nucleoli and mucin were more common in cancer, while cytoplasmic granules, crystalloids and cluster size did not distinguish between PIN and cancer. In conclusion, PIN should not be diagnosed by FNAC alone. However, a highly cellular smear with pronounced atypia seems to preclude PIN.

Switch to full-length of XAF1 mRNA expression in prostate cancer cells by the DNA methylation inhibitor.

X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptotic cell death by binding to caspases and inhibiting their functions, while the XIAP-associated factor1 (XAF1), a zinc finger protein, antagonizes XIAP activities, thereby promoting apoptosis. The aberrant silence of the XAF1 gene has recently been found in various types of cancer cells, which is suggested to be one of the potential mechanisms underlying survival advantages of malignant cells. In the present study, we investigated the XAF1 expression in prostate cancer cells. Compared with normal tissues where a full-length of XAF1 mRNA is predominant, LNCaP and DU145 prostate cancer cell lines only expressed a short form of XAF1 transcripts, whereas PC3 cells exhibited a complete silence of the XAF1 gene. Inhibition of DNA methylation led to a switch to the full length of XAF1 mRNA expression in LNCaP and DU145 cells. The down-regulation of XAF1 expression was also observed in 6/8 tumor samples derived from patients with prostate cancer. Our findings suggest that splicing alterations or downregulation of the XAF1 transcript may occur during the development of prostate cancers due to the aberrant DNA methylation. The alternative splicing of XAF1 mRNA leads to formation of a truncated XAF1 protein with 19 amino acid deletion in its zinc finger domain, which likely affects its functional interaction with XIAP, and consequently, contributes to the pathogenesis of prostate cancers by disrupting balance of the apoptosis machinery.

Ezrin expression in prostate cancer and benign prostatic tissue.

BACKGROUND: The membrane-linking protein ezrin is highly expressed in several types of human cancers and correlations between its immunoreactivity and histopathological data as well as patient outcome have previously been shown. However, such studies have not yet been done on human prostate cancer. This study assesses ezrin protein expression in a series of clinical specimens. METHODS: Immunohistochemical analysis was used to characterize patterns of ezrin expression in prostatic carcinoma and benign epithelium in 103 radical prostatectomy specimens. Ezrin immunoreactivity (IR) was scored 0 to 3 (absent, weak, moderate or strong staining). Agreement between three independent observers was calculated. RESULTS: Ezrin IR in prostate cancers was moderate or strong in 70% of specimens while negative or only weakly positive in benign epithelium. Interobserver agreement of IR score was substantial (mean weighted kappa 0.70, range 0.67-0.73). Ezrin expression correlated with Gleason score (p=0.016) and seminal vesicle invasion (p=0.006) but not with extraprostatic extension or margin status. No correlation with biochemical recurrence after prostatectomy was found (p=0.19). Urothelial and squamous metaplasia invariably showed moderate or strong ezrin expression. Epithelium of seminal vesicles and ejaculatory ducts was always intensely positive. CONCLUSION: Ezrin was expressed in the majority of prostate cancers and correlated with adverse prognostic factors. Interestingly, high levels of ezrin IR were observed in benign metaplastic epithelium and in seminal vesicles.

Prognostic value of serum markers for prostate cancer.

The incidence of prostate cancer has increased dramatically during the last 10-15 years and it is now the commonest cancer in males in developed countries. The increase is mainly caused by the increasing use of opportunistic screening or case-finding based on the use of prostate-specific antigen (PSA) testing in serum. With this approach, prostate cancer is detected 5-10 years before giving rise to symptoms and on average 17 years before causing the death of the patient. While this has led to detection of prostate cancer at a potentially curable stage, it has also led to substantial overdiagnosis, i.e. detection of cancers that would not surface clinically in the absence of screening. A major challenge is thus to identify the cases that need to be treated while avoiding diagnosing patients who will not benefit from being diagnosed and who will only suffer from the stigma of being a cancer patient. It would be useful to have prognostic markers that could predict which patients need to be diagnosed and which do not. Ideally, it should be possible to measure these markers using non-invasive techniques, i.e. by means of serum or urine tests. As it is very useful for both early diagnosis and monitoring of prostate cancer, PSA is considered the most valuable marker available for any tumor. Although the prognostic value of PSA is limited, measurement of the proportion of free PSA has improved the identification of patients with aggressive disease. Furthermore, the rate of increase in serum PSA reflects tumor growth rate and prognosis but, due to substantial physiological variation in serum PSA, reliable estimation of the rate of PSA increase requires follow-up for at least 2 years. Algorithms based on the combined use of free and total PSA and prostate volume in logistic regression and neural networks can improve the diagnostic accuracy for prostate cancer, and assays for minor subfractions of PSA and other new markers may provide additional prognostic information. Markers of neuroendocrine differentiation are useful for the monitoring of androgen-independent disease and various bone markers are useful in patients with metastatic disease.

Differential expression of full-length telomerase reverse transcriptase mRNA and telomerase activity between normal and malignant renal tissues.

Activation of telomerase, a key event during immortalization and malignant transformation, requires expression of the telomerase reverse transcriptase (hTERT). Consistently, lack of telomerase activity and hTERT expression occurs in most normal human somatic cells. However, it has been observed that both normal and cancerous renal tissues express hTERT whereas only the latter exhibits telomerase activity. The mechanism underlying the dissociation between hTERT expression and telomerase activity is unclear. In the present study, we examined telomerase activity and alternative splicing of hTERT transcripts in renal cell carcinoma (RCC) specimens and adjacent normal tissues from 33 patients with RCC. Telomerase activity was detectable in 27 of 33 (82%) RCC samples but none in their normal counterparts. Thirty-two of 33 tumors expressed overall hTERT mRNA and 27 of them contained full-length hTERT transcripts, all with telomerase activity. Although 42% (14 of 33) of normal renal samples expressed hTERT mRNA, none of them had full-length hTERT transcripts, coinciding with lack of telomerase activity. The presence of full-length hTERT mRNA and telomerase activity was significantly associated with c-MYC induction. In tumors, absence of full-length hTERT mRNA or telomerase activity defines a subgroup of nonmetastatic, early-stage RCCs. Taken together, telomerase repression in normal renal tissues is attributed to the absence of full-length hTERT transcripts, whereas telomerase activation is achieved via induction of or switch to expression of full-length hTERT mRNA during the oncogenic process of kidneys, and associated with aggressive RCCs.

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine.

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.

Pim-1 expression in prostatic intraepithelial neoplasia and human prostate cancer.

BACKGROUND: PIM-1, an oncogene product of serine/threonine kinase, has been found to play an important role in induction/suppression of apoptosis, cell cycle progression, and transcriptional regulation by phosphorylating the target proteins involved in these processes. Recently, the expression of PIM-1 has been shown to correlate significantly with measures of prostate cancer clinical outcome. METHODS: Immunohistochemical analysis was used to characterize the patterns of PIM-1 expression in high grade prostatic intraepithelial neoplasia (HGPIN) and cancer in 121 radical prostatectomy specimens. RESULTS: Moderate to strong cytoplasmic staining was observed in 68% of cancers, 12% presented nuclear positivity as well. Moderate to strong expression was seen in 76% of tumors with Gleason score (GS) 7 or higher compared to 58% in tumors with GS 6 or lower (P = 0.04). The staining intensity was moderate or strong in 97% of HGPIN lesions. PIM-1 was overexpressed in HGPIN compared to cancer in 65% of cases. Expression in benign glands was negative or only weakly positive. CONCLUSION: Our data suggest that PIM-1 overexpression in HGPIN may be an early event in the development of prostate malignancy. Additionally, PIM-1 expression provides supplementary information for distinguishing HGPIN from benign epithelium.

Expression of ezrin in prostatic intraepithelial neoplasia.

OBJECTIVES: To study the protein expression and gene copy number of ezrin in a set of high-grade prostatic intraepithelial neoplasia (HGPIN) samples with concomitant prostate cancer. Ezrin is a cytoskeleton linker protein that is actively involved in regulating the growth and metastatic capacity of cancer cells. METHODS: Nineteen HGPIN samples obtained from radical prostatectomy specimens were used for the study. Among them, 13 samples also contained invasive prostate cancer. The expression of ezrin was studied by immunohistochemistry. The same samples were also used for fluorescence in situ hybridization to study the gene copy number of ezrin. RESULTS: Immunoreactivity for ezrin was absent or weak in benign prostatic epithelial cells. Weak or moderate immunostaining was detected in 11 of 13 prostate cancer specimens. However, the immunostaining was moderate or strong in all HGPIN samples. In addition, whenever HGPIN and prostate cancer were found in the same sample, the staining was always more intense in the HGPIN cells than in the cancer cells. No alteration was found in the gene copy number detected by fluorescence in situ hybridization. CONCLUSIONS: We have shown that ezrin is overexpressed in HGPIN and prostate cancer compared with adjacent benign prostatic epithelium. In addition, HGPIN has a greater expression level of ezrin compared with that of prostate cancer. Our results indicate that aberrant expression of ezrin might be involved in the pathogenesis of prostate cancer, and ezrin expression may be useful for the diagnosis of HGPIN.

Gene expression profiling identifies clinically relevant subtypes of prostate cancer.

Prostate cancer, a leading cause of cancer death, displays a broad range of clinical behavior from relatively indolent to aggressive metastatic disease. To explore potential molecular variation underlying this clinical heterogeneity, we profiled gene expression in 62 primary prostate tumors, as well as 41 normal prostate specimens and nine lymph node metastases, using cDNA microarrays containing approximately 26,000 genes. Unsupervised hierarchical clustering readily distinguished tumors from normal samples, and further identified three subclasses of prostate tumors based on distinct patterns of gene expression. High-grade and advanced stage tumors, as well as tumors associated with recurrence, were disproportionately represented among two of the three subtypes, one of which also included most lymph node metastases. To further characterize the clinical relevance of tumor subtypes, we evaluated as surrogate markers two genes differentially expressed among tumor subgroups by using immunohistochemistry on tissue microarrays representing an independent set of 225 prostate tumors. Positive staining for MUC1, a gene highly expressed in the subgroups with "aggressive" clinicopathological features, was associated with an elevated risk of recurrence (P = 0.003), whereas strong staining for AZGP1, a gene highly expressed in the other subgroup, was associated with a decreased risk of recurrence (P = 0.0008). In multivariate analysis, MUC1 and AZGP1 staining were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative prostate-specific antigen levels. Our results suggest that prostate tumors can be usefully classified according to their gene expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification.

Mutation analysis of the BRG1 gene in prostate cancer clinical samples.

Previous studies in hereditary and sporadic prostate cancer have indicated the existence of a tumor suppressor gene in chromosomal region 19p13. The BRG1 gene in this region is one of the possible candidates, based on both the frequency of inactivating mutations in human cancer cell lines, including the prostate cancer cell line DU145, and its functional properties. To our knowledge, no studies have been done to evaluate possible involvement of the BRG1 gene in clinical prostate cancer. To accomplish this, we carried out a complete mutation analysis of all 35 BRG1 exons in tumor and constitutional DNA samples from 21 prostate cancer patients. We report the absence of somatic mutations in the panel of samples employed, but the existence of five germline single nucleotide polymorphisms (SNPs) in CpG islands of the BRG1 gene, among them, three novel ones. In conclusion, the study excludes the presence of common BRG1 mutations in prostate cancer.

Difference between Swedish and Japanese men in the association between AR CAG repeats and prostate cancer suggesting a susceptibility-modifying locus overlapping the androgen receptor gene.

Previous studies have shown that short CAG repeats in the androgen receptor (AR) gene are associated with increased risk of prostate cancer. It is unclear if this association is due to linkage disequilibrium with a susceptibility locus or directly linked to the possible functional impact of the length of the CAG repeats. In this study, the number of the AR CAG repeats was determined in prostate cancer patients, benign prostatic hyperplasia (BPH) patients, and controls in both Swedish and Japanese men. Prostate cancer patients included 59 Swedish hereditary, 59 Swedish sporadic and 33 Japanese sporadic cases. BPH patients included 38 Swedish and 33 Japanese cases. Controls included 98 Swedish healthy men and 43 Japanese men without either prostate cancer or BPH. No significant difference in AR CAG repeats was found in comparison between BPH patients and controls. In contrast, both Swedish hereditary and sporadic prostate cancer patients had shorter AR CAG repeats than Swedish controls, but Japanese prostate cancer patients had longer repeats than controls. These differences between the two populations in the association of prostate cancer and the AR CAG repeats may suggest that the AR CAG repeats are in linkage disequilibrium with a prostate cancer susceptibility locus localized in a small region flanking or overlapping the AR gene at Xq12.1.