Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

BACKGROUND: Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. METHODS: Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. RESULTS: Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. CONCLUSIONS: In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer.

Identification of a novel prostate cancer-associated tumor antigen.

BACKGROUND: The identification of antigens that distinguish cancer cells from normal cells is of major importance for the definition of therapeutic targets in human malignancies. Using sera from cancer patients, we have previously reported on the identification of immunologically recognized proteins that belong to the family of cancer testis antigens (CTAs). METHODS: A normal testicular cDNA library was screened with pooled allogeneic sera from patients with prostate cancer using a modified SEREX approach. Subsequently we have identified and characterized a novel antigen, T21, with an expression pattern similar to that of CTAs. mRNA expression of T21 was determined using a panel of whole tissues and prostate cell lines using Q-RT-PCR. For laser microdissection, fresh prostate cancer and benign tissue was obtained using our novel validated harvesting technique. Protein expression and cellular localization of T21 were assessed in prostate cell lines using Western blotting, confocal microscopy and flow cytometry. RESULTS: T21 showed tissue-restricted mRNA expression in gastric, kidney and prostate cancers, and in normal testis and prostate tissues. Following laser microdissection, T21 was significantly over-expressed in malignant compared to benign prostatic epithelium. We have demonstrated expression of T21 at the protein level and confocal microscopy on PC3 cells probed with a T21-monospecific antibody revealed cytoplasmic localization of T21 protein. CONCLUSIONS: The highly restricted expression pattern of T21 makes it an attractive vaccine target for prostate cancer. Several CTAs reportedly induce cytotoxic T-lymphocyte responses, therefore it is reasonable to assume that T21 will be a valuable target for cancer immunotherapy.

EAU guidelines for the management of genitourinary tuberculosis.

Nearly one third of the world's population is estimated to be infected with Mycobacterium tuberculosis. Moreover, tuberculosis is the most common opportunistic infection in AIDS patients. Genitourinary tuberculosis is not very common but it is considered as a severe form of extra-pulmonary tuberculosis The diagnosis of genitourinary tuberculosis is made based on culture studies by isolation of the causative organism; however, biopsy material on conventional solid media may occasionally be required. Drug treatment is the first line therapy in genitourinary tuberculosis. Treatment regimens of 6 months are effective in most of the patients. Although chemotherapy is the mainstay of treatment, surgery in the form of ablation or reconstruction may be unavoidable. Both radical and reconstructive surgery should be carried out in the first 2 months of intensive chemotherapy.

Obtaining fresh prostate cancer tissue for research: a novel biopsy needle and sampling technique for radical prostatectomy specimens.

BACKGROUND: Fresh or fresh-frozen tissue samples are preferred for molecular profiling as formalin fixation degrades intracellular nucleic acids. Radical prostatectomy (RP) specimens are a valuable source of prostate cancer tissue, but the reliance on whole-organ pathological processing for prognostication limits sampling opportunities. Few studies have addressed specific harvesting techniques using prostatectomy specimens. MATERIALS AND METHODS: Ex vivo biopsies were performed on 23 consecutive fresh RP specimens using a purpose-designed needle. A standard sextant approach was used with an additional lateral biopsy on each side. Cores from each lobe were snap-frozen together and sections assessed by a pathologist blinded to the RP and pre-operative biopsy pathology. Comparison with pre-operative biopsies was performed using the t-test and chi(2) statistical tests. Eleven randomly selected RP specimens were further evaluated for the effects of needle tracks and margin perforation. RESULTS: Cancer was detected in 19 of 23 specimens, giving a sensitivity of 83.6%. The average tumor involvement was 28.3% per section compared with 15.6% for pre-operative biopsies (P < 0.02). There was no statistically significant difference between the groups for either Gleason sum score concordance or tumor location concordance. In 3 of 11 cases, needle margin perforation was identified; in none of the cases did it compromise pathological assessment, although in one case a deeper block resection was required. CONCLUSIONS: Ex vivo biopsy is a useful technique for retrieving fresh tissue whilst preserving organ morphology in RP specimens. The purpose-designed needle and harvesting technique provide good yields of cancer tissue from a high proportion of sampled prostatectomy specimens.

Disabled infectious single cycle herpes simplex virus (DISC-HSV) is a candidate vector system for gene delivery/expression of GM-CSF in human prostate cancer therapy.

BACKGROUND: DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes. METHODS: Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining. RESULTS: Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM. CONCLUSIONS: We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF.