Overexpression of cyclooxygenase-2 in human prostate carcinoma and prostatic intraepithelial neoplasia-association with increased expression of Polo-like kinase-1.

BACKGROUND: Cyclooxygenases (COX) as well as Polo-like kinases (PLK) are involved in proliferation and cell cycle regulation and have been suggested for preventive and therapeutic approaches in prostate carcinoma. METHODS: In this study, we studied expression and prognostic impact of COX-2 in invasive prostate carcinoma, prostatic intraepithelial neoplasia (PIN), atrophic glands, and normal prostatic glands, and investigated the association between COX-2 and PLK-1. RESULTS: We observed a positivity for COX-2 in 72.1% of PIN and in 44.7% of prostate carcinomas with an overexpression of COX-2 in prostate cancer and PIN compared to benign prostatic tissue (P < 0.0005). Furthermore, we observed a strong correlation between expression of PLK-1 and COX-2 (P < 0.0005). CONCLUSIONS: To our knowledge, this is the first report of a correlation between COX-2 and PLK-1 in a malignant tumor. COX-2 and PLK-1 may be interesting targets for new molecular therapies in prostate cancer.

Decreased RECK expression indicating proteolytic imbalance in prostate cancer is associated with higher tumor aggressiveness and risk of prostate-specific antigen relapse after radical prostatectomy.

OBJECTIVES: Decreased expression of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) was recently shown in several cancer types. To evaluate its potential role for prostate carcinoma, we investigated RECK expression in prostate cancer (pCA) samples. METHODS: RECK messenger RNA levels in 15 microdissected normal/tumor matches were determined by quantitative reverse transcriptase-polymerase chain reaction. Protein expression of RECK was evaluated by immunohistochemical staining in tissue samples of adenomectomies (n=24) and pCA samples after radical prostatectomy (n=247). RECK expression was related to preoperative prostate-specific antigen (PSA), tumor stage and grade, surgical margin status, and PSA relapse-free time after radical prostatectomy. RESULTS: Consistent with lower RECK messenger RNA by 24%, RECK protein expression was decreased in pCA, compared with adjacent normal tissue and prostatic intraepithelial neoplasia. RECK expression in samples of benign prostatic hyperplasia from adenomectomy specimens was higher than in normal adjacent tissue of prostate carcinomas. Decreased RECK expression was associated with higher Gleason score (> or =7) and higher tumor stage. Multivariate analysis using the Cox proportional hazards model revealed that negative RECK expression was an independent prognostic factor for an increased risk of PSA relapse, especially in patients with higher tumor grades (Gleason score > or =7). CONCLUSIONS: Decreased RECK expression correlating with the aggressiveness of pCA and the PSA relapse-free time could become an adjunct tissue biomarker to improve the follow-up and treatment decision for these pCA patients.

Tissue pretreatment with formic acid might lower HercepTest scores in breast cancer.

Creutzfeldt-Jakob disease and other prion diseases are diseases with yet not well-defined routes of transmission and infection. The safe processing of potentially contaminated tissue material remains a challenge for histologic laboratories. Formic acid pretreatment is considered to be effective in prion inactivation. We evaluated the c-erbB2 and the hormone receptor-status in potentially prion infectious breast cancer tissue after pretreatment with formic acid. Paired breast cancer tissue samples were immunostained with commercially available antibodies against c-erbB2, estrogen receptor, and progesterone receptor with 1 tissue sample of each pair being pretreated with 98% formic acid. Staining was evaluated either according to the HercepTest score or using an immunoreactive score. Additionally, fluorescence in situ hybridization (FISH) analyses were performed for 7 of these cases. Untreated tissues showed strong circumferential staining for c-erbB2 (HercepTest score 3+), whereas the membranous staining of the tissues pretreated with formic acid was significantly weaker. FISH analyses showed no differences in both groups. The hormone receptor expression was not significantly influenced and positivity was maintained in all cases. In breast cancer patients, the pretreatment of tissue with formic acid for prion-decontamination in the case of suspected Creutzfeldt-Jakob disease or other prion diseases can lead to underestimation of the immunohistologically determined c-erbB2 status. In these cases, a c-erbB2-FISH analysis should be performed. For the immunostaining of hormone receptors in breast cancer, formic acid pretreatment can be applied without negative effects on the sensitivity or specificity of the assay.

Anaplastic large-cell non-Hodgkin's lymphoma of the breast in periprosthetic localisation 32 years after treatment for primary breast cancer--a case report.

Primary, as well as secondary, lymphomas of the breast are rare diseases and might, in some cases, be misdiagnosed as breast cancer on routine hematoxylin/eosin stainings. We report a case of an anaplastic large cell lymphoma in a 72-year-old woman with a history of breast cancer treated with breast-ablative surgery and a subsequent silicon implant 32 years ago. Clinically, she presented with an ulceration of the skin, which had developed within a few months. On conventional histology, the tumor cells were mimicking poorly differentiated invasive ductal carcinoma with a prominent leukocytic infiltrate. The immunoprofile of the tumor showed negativity for cytokeratins and led to the diagnosis of a CD30-positive anaplastic large cell lymphoma.

Clinical value of vesical leukoplakia and evaluation of the neoplastic risk by mutation analyses of the tumor suppressor gene TP53.

AIM: Leukoplakia has been found to be precancerous in organs covered with squamous epithelium. The present study was conducted to determine whether leukoplakia described in the female bladder is also a premalignant lesion. METHODS: Between 1973 and 1996, 77 female patients were diagnosed with vesical leukoplakia by cystoscopy and cytology and were followed-up until 2004 (mean follow-up time: 8.3 years). A survey was conducted to analyze exposure to cocarcinogens. Additionally, DNA was isolated from 36 urine sediments and analyzed for TP53 mutations. The results were compared to the mutation frequency of TP53 in urine sediments from patients diagnosed with transitional cell carcinoma (TCC) of the bladder and healthy controls. RESULTS: The whitish lesion was mostly located at the trigone and varied in size and location during the follow-up years. TP53 mutations were detected in 6 out of 36 urine samples in exons 5, 6 and 7 (mutation frequency: 16.7%). Among control patients with no leukoplakia or TCC of the bladder (n = 70), the spontaneous mutation frequency was similar (14.3%). In contrast, the mutation frequency in patients with TCC of the bladder (n = 148) revealed 39.9% in exons 5, 6, 7 and 8. The present study did not show any statistically significant correlations between chronic inflammations, TP53 mutations, exposure to carcinogens and vesical leukoplakia. CONCLUSIONS: Our data suggest that vesical leukoplakia does not necessarily hold neoplastic potential and needs to be clearly distinguished from leukoplakia in other localizations. Therefore, we suggest that a biopsy can be omitted, if follow-up controls by cystoscopy are performed regularly.

Prognostic relevance of AGR2 expression in breast cancer.

PURPOSE: We aimed to evaluate the expression of the human anterior gradient-2 (AGR2) in breast cancer on RNA and protein level and to correlate it with clinicopathologic data, including patient survival. EXPERIMENTAL DESIGN: AGR2 mRNA expression was assessed by reverse transcription-PCR in 25 breast cancer samples and normal tissues. A polyclonal rabbit AGR antiserum was used for immunohistochemistry on 155 clinicopathologically characterized cases. Statistical analyses were applied to test for prognostic and diagnostic associations. RESULTS: Immunohistochemical detection of AGR2 was statistically significantly associated with positive estrogen receptor status and lower tumor grade. AGR2-positive tumors showed significantly longer overall survival times in univariate analyses. For the subgroup of nodal-negative tumors, an independent prognostic value of AGR2 was found. CONCLUSIONS: The expression of AGR2 in breast cancer is strongly associated with markers of tumor differentiation (estrogen receptor positivity, lower tumor grade). A prognostic effect of AGR2 for overall survival could be shown, which became independently significant for the group of nodal-negative tumors.

Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?

Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and >or=7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.

Cytoplasmic CD24 expression in colorectal cancer independently correlates with shortened patient survival.

PURPOSE: CD24 is a cell adhesion molecule that has been implicated in metastatic tumor progression of various solid tumors. We aimed to clarify the expression patterns of CD24 in colorectal cancer and to correlate these to clinicopathologic variables including patient survival. EXPERIMENTAL DESIGN: 147 colorectal carcinomas and two colon carcinoma cell lines were immunostained for CD24. Cytoplasmic and membranous immunoreactivity were semiquantitatively scored. Fisher's exact test, chi(2) test for trends, Kaplan-Meier analysis, and Cox's regression were applied. RESULTS: The cell line CX-2 showed only a minimal membranous CD24 immunoreactivity, in contrast to HT29, which stained strongly in the cytoplasm. In colorectal cancer, 68.7% of the tumors showed membranous CD24 staining, whereas 84.4% showed cytoplasmic staining. In 10% of cases, an exceptionally strong cytoplasmic CD24 expression was observed. The latter significantly correlated to higher tumor stages (Dukes and pT), nodal or systemic metastasis, and higher tumor grade. In survival analysis, strong cytoplasmic CD24 expression correlated significantly (Cox's regression: P = 0.012, relative risk = 3.7) to shortened patient survival in the group of cases without distant metastases. CONCLUSIONS: CD24 is commonly up-regulated in colorectal cancer and is a new independent prognostic marker which corroborates the importance of CD24 in tumor progression of this disease.

Increased CD59 protein expression predicts a PSA relapse in patients after radical prostatectomy.

BACKGROUND: Human protectin (CD59) is a regulator of complement activation that inhibits complement-mediated cell lysis, and thus might confer immune resistance to tumor cells. CD59 expression has been described in a variety of human malignancies, including breast cancer. Since a comprehensive investigation of CD59 expression in prostate cancer has not been conducted yet, we aimed to determine the significance of CD59 expression in prostate cancer. METHODS: Eighty-six primary adenocarcinomas of the prostate were immunostained using a monoclonal CD59 antibody (clone MEM-43) and a standard detection system. The immunoreactivity of the tumor was evaluated as low versus high for statistical analysis. Additionally, CD59 mRNA levels were determined by real-time PCR in matched (tumor/normal) microdissected tissues from 26 cases. RESULTS: Cytoplasmic CD59 immunoreactivity was found in epithelia of prostate cancer, prostatic intraepithelial neoplasia, benign hyperplasia, atrophic, and normal glands. High rates of CD59 expression were noted in 36% of prostate cancer cases and were significantly associated with tumor pT stage (P = 0.043), Gleason grade (P = 0.013) and earlier biochemical (PSA) relapse in Kaplan-Meier analysis (P = 0.0013). On RNA level, we found an upregulation in 19.2% (five cases), although the general rate of CD59 transcript was significantly lower in tumor tissue (P = 0.03). CONCLUSION: CD59 protein is strongly expressed in 36% of adenocarcinomas of the prostate and and is associated with disease progression and adverse patient prognosis.

Endothelium-dependent and -independent relaxation and VASP serines 157/239 phosphorylation by cyclic nucleotide-elevating vasodilators in rat aorta.

Endothelium-dependent vasodilation is thought to be mediated primarily by the NO/cGMP signaling pathway whereas cAMP-elevating vasodilators are considered to act independent of the endothelial cell layer. However, recent functional data suggest that cAMP-elevating vasodilators such as beta-receptor agonists, adenosine or forskolin may also be endothelium-dependent. Here we used functional and biochemical assays to analyze endothelium-dependent, cGMP- and cAMP-mediated signaling in rat aorta. Acetylcholine and sodium nitroprusside (SNP) induced a concentration-dependent relaxation of phenylephrine-precontracted aorta. This response was reflected by the phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a validated substrate of cGMP- and cAMP-dependent protein kinases (cGK, cAK), on Ser(157) and Ser(239). As expected, the effects of acetylcholine were endothelium-dependent. However, relaxation induced by the beta-receptor agonist isoproterenol was also almost completely impaired after endothelial denudation. At the biochemical level, acetylcholine- and isoproterenol-evoked cGK and cAK activation, respectively, as measured by VASP Ser(239) and Ser(157) phosphorylation, was strongly diminished. Furthermore, the effects of isoproterenol were repressed by eNOS inhibition when endothelium was present. We also observed that the relaxing and biochemical effects of forskolin were at least partially endothelium-dependent. We conclude that cAMP-elevating vasodilators, i.e. isoproterenol and to a lesser extent also forskolin, induce vasodilation and concomitant cyclic nucleotide protein kinase activation in the vessel wall in an endothelium-dependent way.